Semiconductor nanocrystal probes for biological applications and process for making and using such probes

ABSTRACT

A semiconductor nanocrystal compound is described which is capable of linking to one or more affinity molecules. The compound comprises (1) one or more semiconductor nanocrystals capable of, in response to exposure to a first energy, providing a second energy, and (2) one or more linking agents, having a first portion linked to the one or more semiconductor nanocrystals and a second portion capable of linking to one or more affinity molecules. One or more of these semiconductor nanocrystal compounds are linked to one or more affinity molecules to form a semiconductor nanocrystal probe capable of bonding with one or more detectable substances in a material being analyzed, and capable of, in response to exposure to a first energy, providing a second energy. In one embodiment, the probe is capable of emitting electromagnetic radiation in a narrow wavelength band and/or absorbing, scattering, or diffracting energy when excited by an electromagnetic radiation source (of narrow or broad bandwidth) or a particle beam. The probe is stable to repeated exposure to energy in the presence of oxygen and/or other radicals.  
     Treatment of a material with the semiconductor nanocrystal probe, and subsequent exposure of this treated material to a first energy, to determine the presence of the detectable substance within the material bonded to the probe, will excite the semiconductor nanocrystal in the probe bonded to the detectable substance, causing the probe to provide a second energy signifying the presence, in the material, of the detectable substance bonded to the semiconductor nanocrystal probe. In one embodiment, the semiconductor nanocrystals in the probe are excitable over a broad bandwidth of energy, and emit electromagnetic radiation over a narrow bandwidth, making it possible to use a single energy source to simultaneously excite a plurality of such probes, each emitting electromagnetic radiation of a differing wavelength band to simultaneously analyze for a plurality of detectable substances in a material being analyzed.  
     Also described are processes for respectively making the semiconductor nanocrystal compound and the semiconductor nanocrystal probe. Processes are also described for treating materials with the probe, for example, to determine the presence of a detectable substance in the material bonded to the probe.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation in part of U.S. Patentapplication Ser. No. 08/978,450 filed Nov. 25, 1997, and assigned to theassignee of this application.

BACKGROUND OF THE INVENTION

[0002] The invention described herein arose in the course of, or under,Contract No. DE-AC03-SF00098 between the United States Department ofEnergy and the University of California for the operation of the ErnestOrlando Lawrence Berkeley National Laboratory. The Government may haverights to the invention.

[0003] 1. Field of the Invention

[0004] This invention relates to semiconductor nanocrystal probes forbiological applications wherein the probes include a plurality ofsemiconductor nanocrystals capable of providing a detectable signal inresponse to exposure to energy.

[0005] 2. Description of the Related Art

[0006] Fluorescent labeling of biological systems is a well knownanalytical tool used in modern biotechnology as well as analyticalchemistry. Applications for such fluorescent labeling includetechnologies such as medical (and non-medical) fluorescence microscopy,histology, flow cytometry, fluorescence in-situ hybridization (medicalassays and research), DNA sequencing, immuno-assays, binding assays,separation, etc.

[0007] Conventionally, such fluorescent labeling involves the use of anorganic dye molecule bonded to a moiety which, in turn, selectivelybonds to a particular biological system, the presence of which is thenidentified by excitation of the dye molecule to cause it to fluoresce.There are a number of problems with such an analytical system. In thefirst place, the emission of light of visible wavelengths from anexcited dye molecule usually is characterized by the presence of a broademission spectrum as well as a broad tail of emissions on the red sideof the spectrum, i.e., the entire emission spectrum is rather broad. Asa result, there is a severe limitation on the number of different colororganic dye molecules which may be utilized simultaneously orsequentially in an analysis since it is difficult to eithersimultaneously or even non-simultaneously detect or discriminate betweenthe presence of a number of different detectable substances due to thebroad spectrum emissions and emission tails of the labeling molecules.Another problem is that most dye molecules have a relatively narrowabsorption spectrum, thus requiring either multiple excitation beamsused either in tandem or sequentially for multiple wavelength probes, orelse a broad spectrum excitation source which is sequentially used withdifferent filters for sequential excitation of a series of probesrespectively excited at different wavelengths.

[0008] Another problem frequently encountered with existing dye moleculelabels is that of photostability. Available fluorescent moleculesbleach, or irreversibly cease to emit light, under repeated excitation(10⁴-10⁸ cycles of absorption/emission). These problems are oftensurmounted by minimizing the amount of time that the sample is exposedto light, and by removing oxygen and/or other radical species from thesample.

[0009] In addition, the probe tools used for the study of systems byelectron microscopy techniques are completely different from the probesused for study by fluorescence. Thus, it is not possible to label amaterial with a single type of probe for both electron microscopy andfor fluorescence.

[0010] It would, therefore, be desirable to provide a stable probematerial for biological applications preferably having a wide absorptionband and capable of providing a detectable signal in response toexposure to energy, without the presence of the large red emission tailscharacteristic of dye molecules (thereby permitting the simultaneous useof a number of such probe materials, each, for example, emitting lightof a different narrow wavelength band) and/or capable of scattering ordiffracting radiation. It would also be equally desirable to provide asingle, stable probe material which can be used to image the same sampleby both light and electron microscopy.

SUMMARY OF THE INVENTION

[0011] The invention comprises a semiconductor nanocrystal compoundcapable of linking to one or more affinity molecules to form asemiconductor nanocrystal probe. The semiconductor nanocrystal compoundcomprises one or more semiconductor nanocrystals and one or more firstlinking agents. The one or more semiconductor nanocrystals are capableof providing a detectable signal in response to exposure to energy,wherein such a response may include emission and/or absorption and/orscattering or diffraction of the energy to which the one or moresemiconductor nanocrystals are exposed. In addition to or as analternative to providing a detectable signal, the one or moresemiconductor nanocrystals may transfer energy to one or more proximalstructures in response to exposure to energy. The one or more firstlinking agents have a first portion linked to one or more semiconductornanocrystals and a second portion capable of linking either to one ormore second linking agents or to one or more affinity molecules.

[0012] The invention further comprises a semiconductor nanocrystal probeformed either by (1) linking one or more of the above describedsemiconductor nanocrystal compounds to one or more affinity molecules;or (2) linking one or more of the above described semiconductornanocrystal compounds to one or more second linking agents and linkingthe one or more second linking agents to one or more affinity molecules,wherein the one or more affinity molecules are capable of bonding to oneor more detectable substances in a material. As a result, thesemiconductor nanocrystal probe, in one embodiment, is capable ofabsorbing energy from either a particle beam or an electromagneticradiation source (of broad or narrow bandwidth), and is capable ofemitting detectable electromagnetic radiation in a narrow wavelengthband when so excited; while in another embodiment the amount of energyfrom either a particle beam or an electromagnetic radiation source (ofbroad or narrow bandwidth) which is absorbed, or scattered, ordiffracted by the semiconductor nanocrystal probe, is detectable, i.e.,the change in absorption, scattering, or diffraction is detectable. Inyet another embodiment, the semiconductor nanocrystal probe is capableof receiving energy transferred from a proximal source and/ortransferring energy to one or more proximal structures in response toexposure to energy.

[0013] The invention also comprises a process for making thesemiconductor nanocrystal compound and for making the semiconductornanocrystal probe comprising the semiconductor nanocrystal compoundlinked to one or more affinity molecules capable of bonding to one ormore detectable substances. The semiconductor nanocrystal probe of theinvention is stable with respect to repeated excitation by light, orexposure to elevated temperatures, or exposure to oxygen or otherradicals.

[0014] The invention further comprises a process for treating amaterial, such as a biological material, to determine the presence of adetectable substance in the material, which comprises a step ofcontacting the material to be treated, with the semiconductornanocrystal probe, an optional step of removing from the material thesemiconductor nanocrystal probes not bonded to the detectable substance,and then a step of exposing the material to energy from, for example,either an electromagnetic radiation source (of broad or narrowbandwidth) or a particle beam. The presence of the detectable substancein the material is then determined by a step of detecting the signalprovided by the semiconductor nanocrystal probe in response to exposureto energy. This may be accomplished, for example, either by measuringthe absorption of energy by the semiconductor nanocrystal probe and/ordetecting the emission of radiation of a narrow wavelength band by thesemiconductor nanocrystal probe and/or detecting the scattering ordiffraction of energy by the semiconductor nanocrystal probe, indicative(in either case) of the presence of the semiconductor nanocrystal probebonded to the detectable substance in the material.

[0015] The invention further comprises a process for treating amaterial, such as a biological material with a semiconductor nanocrystalprobe which is used to transfer energy to one or more proximalstructures. This process comprises a step of contacting the material tobe treated, with the semiconductor nanocrystal probe, an optional stepof removing from the material portions of the semiconductor nanocrystalprobe not bonded to the detectable substance, and then a step ofexposing the material to energy from, for example, either anelectromagnetic radiation source (of broad or narrow bandwidth) or aparticle beam. This is followed by a step of energy transfer from thesemiconductor nanocrystal probe to one or more proximal structures whichmay, in response to the energy transfer, either provide a detectablesignal, undergo chemical or conformational changes, or transfer energyto one or more second proximal structures.

[0016] The use of the semiconductor nanocrystal probe in the treatmentof a material to either provide a detectable signal or transfer energyto a proximal structure may be applied to a plurality of medical andnon-medical biological applications. Exemplary applications of thesemiconductor nanocrystal probe include: use as a detector of substanceson the surface or interior of cells in flow cytometry; use in aplurality of methods for detecting nucleic acid sequences byhybridization, such as fluorescence in-situ hybridization (particularlywhen the semiconductor nanocrystal probe has been modified in apolymerase chain reaction); or use to transfer energy which may causethe release of a cytotoxic molecule or transfer of heat energy, eitherof which may result in death of specifically targeted cells. Another useof the semiconductor nanocrystal probe is as a precursor which istreated to synthetic steps which result in a modified semiconductornanocrystal probe (as in the case of modification by polymerase chainreaction).

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a block diagram of the semiconductor nanocrystalcompound of the invention.

[0018]FIG. 2 is a block diagram of the semiconductor nanocrystal probeof the invention.

[0019]FIG. 3 is a block diagram showing the affinity between adetectable substance and the semiconductor nanocrystal probe of theinvention.

[0020]FIG. 4 is a flow sheet illustrating the process of forming thesemiconductor nanocrystal probe of the invention.

[0021]FIG. 5 is a flow sheet illustrating a typical use of thesemiconductor nanocrystal probe of the invention in detecting thepresence of a detectable substance in a material such as a biologicalmaterial.

[0022]FIG. 6 is a flow sheet illustrating a typical use of thesemiconductor nanocrystal probe of the invention in transferring energyto a proximal structure.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The invention comprises a semiconductor nanocrystal compoundcapable of linking to either one or more second linking agents or to oneor more affinity molecules, and capable of providing a detectable signalin response to exposure to energy. The semiconductor nanocrystalcompound, in turn, comprises: (1) one or more semiconductornanocrystals, each capable of providing a detectable signal in responseto exposure to energy; and (2) one or more first linking agents, eachhaving a first portion linked to the semiconductor nanocrystal and asecond portion capable of linking either to one or more second linkingagents or to one or more affinity molecules.

[0024] The invention also comprises the above described semiconductornanocrystal compound linked to one or more affinity molecules (througheither one or more first linking agents, or through one or more secondlinking agents which are in turn linked to one or more first linkingagents) to form a semiconductor nanocrystal probe capable of bonding toone or more detectable substances and capable of providing a detectablesignal in response to exposure to energy. Treatment of a material(typically a biological material) with the semiconductor nanocrystalprobe, and subsequent exposure of this treated material to energy, asdescribed above, to determine the presence of the detectable substancewithin the material, will result in the semiconductor nanocrystal in thesemiconductor nanocrystal probe bonded to the detectable substanceproviding a detectable signal. This detectable signal, such as a changein absorption and/or emission of electromagnetic radiation of a narrowwavelength band and/or scattering or diffraction may signify (in eitherinstance) the presence in the material, of the detectable substancebonded to the semiconductor nanocrystal probe.

[0025] The invention also comprises a process for making thesemiconductor nanocrystal compound, and a process for making thesemiconductor nanocrystal probe comprising the semiconductor nanocrystalcompound linked to one or more affinity molecules capable of bonding toone or more detectable substances.

[0026] The invention further comprises a process for treating amaterial, such as a biological material, to determine the presence ofone or more detectable substances in the material which comprises: (1)contacting the material with the semiconductor nanocrystal probe, (2)(optionally) removing from the material the semiconductor nanocrystalprobes not bonded to the detectable substance, (3) exposing the materialto energy (such as the above-described electromagnetic energy source orparticle beam), to which, the semiconductor nanocrystal is capable ofproviding a response, signifying the presence of the semiconductornanocrystal probe bonded to the detectable substance in the material,and (4) detecting the signal provided by the semiconductor nanocrystalin the semiconductor nanocrystal probe.

[0027] The invention further comprises a process for treating amaterial, such as a biological material, using a semiconductornanocrystal probe, which comprises: (1) contacting the material with thesemiconductor nanocrystal probe, (2) (optionally) removing from thematerial the semiconductor nanocrystal probes not bonded to thedetectable substance, (3) exposing the material to energy (such as anelectromagnetic energy source or particle beam) capable of causing atransfer of energy from one or more semiconductor nanocrystal probes toone or more proximal structures in response to exposure to energy, and(4) transferring energy from one or more semiconductor nanocrystalprobes to one or more proximal structures.

a. Definitions

[0028] By use of the terms “nanometer crystal” or “nanocrystal” hereinis meant an organic or inorganic crystal particle, preferably a singlecrystal particle, having an average cross-section no larger than about20 nanometers (nm) or 20×10⁻⁹ meters (200 Angstroms), preferably nolarger than about 10 nm (100 Angstroms) and a minimum averagecross-section of about 1 nm, although in some instances a smalleraverage cross-section nanocrystal, i.e., down to about 0.5 nm (5Angstroms), may be acceptable. Typically the nanocrystal will have anaverage cross-section ranging in size from about 1 nm (10 Angstroms) toabout 10 nm (100 angstroms).

[0029] By use of the term “semiconductor nanocrystal” is meant ananometer crystal or nanocrystal of Group II-VI and/or Group III-Vsemiconductor compounds capable of emitting electromagnetic radiationupon excitation, although the use of Group IV semiconductors such asgermanium or silicon, or the use of organic semiconductors, may befeasible under certain conditions.

[0030] The term “radiation,” as used herein, is meant to includeelectromagnetic radiation, including x-ray, gamma, ultra-violet,visible, infra-red, and microwave radiation; and particle radiation,including electron beam, beta, and alpha particle radiation.

[0031] The term “energy” is intended to include electromagneticradiation, particle radiation, and fluorescence resonance energytransfer (FRET). As used herein, the term “first energy” is meant theenergy to which a semiconductor nanocrystal is exposed. Use of the term“second energy” is meant energy provided by a semiconductor nanocrystal,within a semiconductor nanocrystal compound or within a semiconductornanocrystal probe, in response to exposure to a first energy. It shouldbe noted that different nanocrystals, when exposed to the same “firstenergy”, may respectively provide “second energies” which differ fromone another, and the use of the term “second energy”, when used inconnection with a plurality of semiconductor nanocrystals will beunderstood to refer to either second energies which are the same or to aplurality of different second energies.

[0032] By the use of the term “energy transfer” is meant the transfer ofenergy from one atom or molecule to another atom or molecule by eitherradiative or non-radiative pathways.

[0033] The term “proximal source” is meant an atom, a molecule, or anyother substance which is capable of transferring energy to and/orreceiving energy transferred from another atom or molecule or any othersubstance.

[0034] The term “proximal structure” as used herein may be an atom, amolecule, or any other substance (e.g. a polymer, a gel, a lipidbilayer, and any substance bonded directly to a semiconductornanocrystal probe) which is capable of receiving energy transferred fromanother atom or molecule or other substance (including a semiconductornanocrystal probe).

[0035] By use of the term “a narrow wavelength band”, with regard to theelectromagnetic radiation emission of the semiconductor nanocrystal, ismeant a wavelength band of emissions not exceeding about 40 nm, andpreferably not exceeding about 20 nm in width and symmetric about thecenter, in contrast to the emission bandwidth of about 100 nm for atypical dye molecule, with a red tail which may extend the band widthout as much as another 100 nm. It should be noted that the bandwidthsreferred to are determined from measurement of the width of theemissions at half peak height (FWHM), and are appropriate in the rangeof 200 nm to 2000 nm.

[0036] By use of the term “a broad wavelength band”, with regard to theelectromagnetic radiation absorption of the semiconductor nanocrystal ismeant absorption of radiation having a wavelength equal to, or shorterthan, the wavelength of the onset radiation (the onset radiation isunderstood to be the longest wavelength (lowest energy) radiationcapable of being absorbed by the semiconductor nanocrystal), whichoccurs near to, but at slightly higher energy than the “narrowwavelength band” of the emission. This is in contrast to the “narrowabsorption band” of dye molecules which occurs near the emission peak onthe high energy side, but drops off rapidly away from that wavelengthand is often negligible at wavelengths further than 100 nm from theemission.

[0037] The term “detectable signal,” as used herein, is meant to includeemission by the semiconductor nanocrystal of electromagnetic radiation,including visible or infrared or ultraviolet light and thermal emission;and any other signal or change in signal emanating from thesemiconductor nanocrystal evidencing scattering (including diffraction)and/or absorption in response to exposure of the semiconductornanocrystal to radiation.

[0038] By use of the term “detectable substance” is meant an entity orgroup or class of groups, the presence or absence of which, in amaterial such as a biological material, is to be ascertained by use ofthe semiconductor nanocrystal probe of the invention.

[0039] By use of the term “affinity molecule” is meant the portion ofthe semiconductor nanocrystal probe of the invention which comprises anatom, molecule, or other moiety capable of selectively bonding to one ormore detectable substances (if present) in the material (e.g.,biological material) being analyzed.

[0040] The use of the term “small molecule” as used herein (for eitheran affinity molecule or a detectable substance) is any atom or molecule,inorganic or organic, including biomolecules, having a molecular weightbelow about 10,000 daltons (grams/mole).

[0041] By use of the term “linking agent” is meant a substance capableof linking with one or more semiconductor nanocrystals and also capableof linking to one or more affinity molecules or one or more secondlinking agents.

[0042] By use of the term “first linking agent” is meant a substancecapable of either (1) linking with one or more semiconductornanocrystals, and also capable of linking to one or more affinitymolecules; or (2) linking with one or more semiconductor nanocrystalsand also capable of linking to one or more second linking agents.

[0043] By use of the term “second linking agent” is meant a substancecapable of linking to one or more affinity molecules and also capable oflinking to one or more linking agents.

[0044] Use of the term “three-dimensional structure” herein is meant todefine any structure, independent of shape, which is greater than 10 nmin thickness along the three mutually perpendicular principle axes ofthe structure.

[0045] Use of the term “substructure” herein is meant one of two or moreportions of a three-dimensional structure.

[0046] The terms “link” and “linking” are meant to describe theadherence between the one or more affinity molecules and the one or moresemiconductor nanocrystals, either directly or through one or moremoieties identified herein as linking agents (including second linkingagents between the linking agent and the affinity molecule). Theadherence may comprise any sort of bond, including, but not limited to,covalent, ionic, hydrogen bonding, van der Waals forces, or mechanicalbonding, etc.

[0047] The terms “bond” and “bonding” are meant to describe theadherence between the affinity molecule and the detectable substance.The adherence may comprise any sort of bond, including, but not limitedto, covalent, ionic, or hydrogen bonding, van der Waals forces, ormechanical bonding, etc.

[0048] The term “semiconductor nanocrystal compound”, as used herein, isintended to define one or more semiconductor nanocrystals linked to oneor more first linking agents and capable of linking to either one ormore second linking agents or to one or more affinity molecules, whilethe term “semiconductor nanocrystal probe” is intended to define asemiconductor nanocrystal compound linked to one or more affinitymolecules.

[0049] The term “glass” as used herein is intended to include one ormore oxides of silicon, boron, and/or phosphorus, or a mixture thereof,as well as the further optional inclusion of one or more metalsilicates, metal borates or metal phosphates therein.

b. The Semiconductor Nanocrystals

[0050] The semiconductor nanocrystals useful in the practice of theinvention include nanocrystals of Group II-VI semiconductors such asMgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS,ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, and HgTe as well as mixedcompositions thereof; as well as nanocrystals of Group III-Vsemiconductors such as GaAs, InGaAs, InP, and InAs and mixedcompositions thereof. As mentioned above, the use of Group IVsemiconductors such as germanium or silicon, or the use of organicsemiconductors, may also be feasible under certain conditions. Thesemiconductor nanocrystals may also include alloys comprising two ormore semiconductors selected from the group consisting of the aboveGroup III-V compounds, Group II-VI compounds, Group IV elements, andcombinations of same.

[0051] Formation of nanometer crystals of Group III-V semiconductors isdescribed in copending and commonly assigned Alivisatos et al. U.S. Pat.No. 5,571,018; Alivisatos et al. U.S. Pat. No. 5,505,928; and Alivisatoset al. U.S. Pat. No. 5,262,357, which also describes the formation ofGroup II-VI semiconductor nanocrystals, and which is also assigned tothe assignee of this invention. Also described therein is the control ofthe size of the semiconductor nanocrystals during formation usingcrystal growth terminators. The teachings of Alivisatos et al. U.S. Pat.No. 5,571,018, and Alivisatos et al. U.S. Pat. No. 5,262,357 are eachhereby specifically incorporated by reference.

[0052] In one embodiment, the nanocrystals are used in a core/shellconfiguration wherein a first semiconductor nanocrystal forms a coreranging in diameter, for example, from about 20 Å to about 100 Å, with ashell of another semiconductor nanocrystal material grown over the corenanocrystal to a thickness of, for example, 1-10 monolayers inthickness. When, for example, a 1-10 monolayer thick shell of CdS isepitaxially grown over a core of CdSe, there is a dramatic increase inthe room temperature photoluminescence quantum yield. Formation of suchcore/shell nanocrystals is described more fully in a publication by oneof us with others entitled “Epitaxial Growth of Highly LuminescentCdSe/CdS Core/Shell Nanocrystals with Photostability and ElectronicAccessibility”, by Peng, Schlamp, Kadavanich, and Alivisatos, publishedin the Journal of the American Chemical Society, Volume 119, No. 30.1997, at pages 7019-7029, the subject matter of which is herebyspecifically incorporated herein by reference.

[0053] The semiconductor nanocrystals used in the invention will have acapability of absorbing radiation over a broad wavelength band. Thiswavelength band includes the range from gamma radiation to microwaveradiation. In addition, these semiconductor nanocrystals will have acapability of emitting radiation within a narrow wavelength band ofabout 40 nm or less, preferably about 20 nm or less, thus permitting thesimultaneous use of a plurality of differently colored semiconductornanocrystal probes with different semiconductor nanocrystals withoutoverlap (or with a small amount of overlap) in wavelengths of emittedlight when exposed to the same energy source. Both the absorption andemission properties of semiconductor nanocrystals may serve asadvantages over dye molecules which have narrow wavelength bands ofabsorption (e.g. about 30-50 nm) and broad wavelength bands of emission(e.g. about 100 nm) and broad tails of emission (e.g. another 100 nm) onthe red side of the spectrum. Both of these properties of dyes impairthe ability to use a plurality of differently colored dyes when exposedto the same energy source.

[0054] Furthermore, the frequency or wavelength of the narrow wavelengthband of light emitted from the semiconductor nanocrystal may be furtherselected according to the physical properties, such as size, of thesemiconductor nanocrystal. The wavelength band of light emitted by thesemiconductor nanocrystal, formed using the above embodiment, may bedetermined by either (1) the size of the core, or (2) the size of thecore and the size of the shell, depending on the composition of the coreand shell of the semiconductor nanocrystal. For example, a nanocrystalcomposed of a 3 nm core of CdSe and a 2 nm thick shell of CdS will emita narrow wavelength band of light with a peak intensity wavelength of600 nm. In contrast, a nanocrystal composed of a 3 nm core of CdSe and a2 nm thick shell of ZnS will emit a narrow wavelength band of light witha peak intensity wavelength of 560 nm.

[0055] A plurality of alternatives to changing the size of thesemiconductor nanocrystals in order to selectably manipulate theemission wavelength of semiconductor nanocrystals exist. Thesealternatives include: (1) varying the composition of the nanocrystal,and (2) adding a plurality of shells around the core of the nanocrystalin the form of concentric shells. It should be noted that differentwavelengths can also be obtained in multiple shell type semiconductornanocrystals by respectively using different semiconductor nanocrystalsin different shells, i.e., by not using the same semiconductornanocrystal in each of the plurality of concentric shells.

[0056] Selection of the emission wavelength by varying the composition,or alloy, of the semiconductor nanocrystal is old in the art. As anillustration, when a CdS semiconductor nanocrystal, having an emissionwavelength of 400 nm, may be alloyed with a CdSe semiconductornanocrystal, having an emission wavelength of 530 nm. When a nanocrystalis prepared using an alloy of CdS and CdSe, the wavelength of theemission from a plurality of identically sized nanocrystals may be tunedcontinuously from 400 nm to 530 nm depending on the ratio of S to Sepresent in the nanocrystal. The ability to select from differentemission wavelengths while maintaining the same size of thesemiconductor nanocrystal may be important in applications which requirethe semiconductor nanocrystals to be uniform in size, or for example, anapplication which requires all semiconductor nanocrystals to have verysmall dimensions when used in application with steric restrictions.

c. Affinity Molecules

[0057] The particular affinity molecule forming a part of thesemiconductor nanocrystal probe of the invention will be selected basedon its affinity for the particular detectable substance whose presenceor absence, for example, in a biological material, is to be ascertained.Basically, the affinity molecule may comprise any molecule capable ofbeing linked to one or more semiconductor nanocrystal compounds which isalso capable of specific recognition of a particular detectablesubstance. In general, any affinity molecule useful in the prior art incombination with a dye molecule to provide specific recognition of adetectable substance will find utility in the formation of thesemiconductor nanocrystal probes of the invention. Such affinitymolecules include, by way of example only, such classes of substances asmonoclonal and polyclonal antibodies, nucleic acids (both monomeric andoligomeric), proteins, polysaccharides, and small molecules such assugars, peptides, drugs, and ligands. Lists of such affinity moleculesare available in the published literature such as, by way of example,the “Handbook of Fluorescent Probes and Research Chemicals”, (sixthedition) by R. P. Haugland, available from Molecular Probes, Inc.

d. The Linking Agents

[0058] The semiconductor nanocrystal probe of the invention will usuallyfind utility with respect to the detection of one or more detectablesubstances in organic materials, and in particular to the detection ofone or more detectable substances in biological materials. This requiresthe presence, in the semiconductor nanocrystal probe, of an affinitymolecule or moiety, as described above, which will bond thesemiconductor nanocrystal probe to the detectable substance in theorganic/biological material so that the presence of the detectablematerial may be subsequently ascertained. However, since thesemiconductor nanocrystals are inorganic, they may not bond directly tothe affinity molecule. In this case therefore, there must be some typeof linking agent present in the semiconductor nanocrystal probe which iscapable of linking the inorganic semiconductor nanocrystal to theaffinity molecule in the semiconductor nanocrystal probe. The linkingagent may be in the form of one or more linking agents linking one ormore semiconductor nanocrystals to one or more affinity molecules.Alternatively, two types of linking agents may be utilized. One or moreof the first linking agents may be linked to one or more semiconductornanocrystals and also linked to one or more second linking agents. Theone or more second linking agents may be linked to one or more affinitymolecules and to one or more first linking agents.

[0059] One form in which the semiconductor nanocrystal may be linked toan affinity molecule via a linking agent is by coating the semiconductornanocrystal with a thin layer of glass, such as silica (SiO_(x), wherex=1-2), using a linking agent such as a substituted silane, e.g.,3-mercaptopropyl-trimethoxy silane to link the nanocrystal to the glass.The glass-coated semiconductor nanocrystal may then be further treatedwith a linking agent, e.g., an amine such as3-aminopropyl-trimethoxysilane, which will function to link theglass-coated semiconductor nanocrystal to the affinity molecule. Thatis, the glass-coated semiconductor nanocrystal may then be linked to theaffinity molecule. It is within the contemplation of this invention thatthe original semiconductor nanocrystal compound may also be chemicallymodified after it has been made in order to link effectively to theaffinity molecule. A variety of references summarize the standardclasses of chemistry which may be used to this end, in particular the“Handbook of Fluorescent Probes and Research Chemicals”, (6th edition)by R. P. Haugland, available from Molecular Probes, Inc., and the book“Bioconjugate Techniques”, by Greg Hermanson, available from AcademicPress, New York.

[0060] When the semiconductor nanocrystal may be coated with a thinlayer of glass, the glass, by way of example, may comprise a silicaglass (SiO_(x), where x=1-2), having a thickness ranging from about 0.5nm to about 10 nm, and preferably from about 0.5 nm to about 2 nm.

[0061] The semiconductor nanocrystal is coated with the coating of thinglass, such as silica, by first coating the nanocrystals with asurfactant such as tris-octyl-phosphine oxide, and then dissolving thesurfactant-coated nanocrystals in a basic methanol solution of a linkingagent, such as 3-mercaptopropyl-tri-methoxy silane, followed by partialhydrolysis which is followed by addition of a glass-affinity moleculelinking agent such as amino-propyl trimethoxysilane which will link tothe glass and serve to form a link with the affinity molecule.

[0062] When the linking agent does not involve the use of a glasscoating on the semiconductor nanocrystal, it may comprise a number ofdifferent materials, depending upon the particular affinity molecule,which, in turn, depends upon the type of detectable material beinganalyzed for. It should also be noted that while an individual linkingagent may be used to link to an individual semiconductor nanocrystal, itis also within the contemplation of the invention that more than onelinking agent may bond to the same semiconductor nanocrystal and viceversa; or a plurality of linking agents may be used to link to aplurality of semiconductor nanocrystals. In addition, when first andsecond linking agents are used, one or more first linking agents may belinked to the same second linking agent, or more than one second linkingagents may be linked to the same first linking agent.

[0063] A few examples of the types of linking agents which may be usedto link to both the semiconductor nanocrystal (or to a glass coating onthe nanocrystal) and to the affinity molecule in the probe areillustrated in the table below, it being understood that this is notintended to be an exhaustive list: Linking Agent Structure Name

N-(3-aminopropyl)3-mercapto-benzamide

3-aminopropyl-trimethoxysilane

3-mercaptopropyl-trimethoxysilane

3-(trimethoxysilyl)propylmaleimide

3-(trimethoxysilyl)propylhydrazide

[0064] It should be further noted that a plurality of polymerizablelinking agents may be used together to form an encapsulating net orlinkage around an individual nanocrystal (or group of nanocrystals).This is of particular interest where the particular linking agent isincapable of forming a strong bond with the nanocrystal. Examples oflinking agents capable of bonding together in such a manner to surroundthe nanocrystal with a network of linking agents include, but are notlimited to: diacetylenes, styrene-butadienes, vinyl acetates, acrylates,acrylamides, vinyl, styryl, and the aforementioned silicon oxide, boronoxide, phosphorus oxide, silicates, borates and phosphates, as well aspolymerized forms of at least some of the above.

e. Compounds and Probes Having Three-Dimensional Structured LinkingAgents

[0065] In one embodiment, the linking agent, including many of thosedescribed above, may be used in, or as, a three-dimensional structurewhich may be either organic or inorganic, and which may be either asolid (porous or non-porous) or hollow. In the prior art, the use of dyemolecules embedded into latex spheres for diagnostic applications iswell established. Perhaps the most common application involvesselectively coloring the latex sphere using one or more dye moleculesand then coating the sphere with a number of proteins of interest.

[0066] The utilization of such a three-dimensional linking agentstructure (which may be most easily conceptualized as a sphere) in thecompound and probe of the invention has the added benefit of permittingsuch a linking agent to have bonded thereto more than one semiconductornanocrystals, as well as one or more affinity molecules (either directlyor through a second linking agent). The three-dimensional linking agentstructure will herein-after be described as a part of a probe(semiconductor nanocrystal, linking agent, and affinity molecule), itbeing understood that the structures described apply to the formation ofa compound (semiconductor nanocrystal and linking agent) as well as aprobe.

[0067] The advantage of a compound or probe in which one or moresemiconductor nanocrystals are bonded to a three-dimensional linkingagent structure lies in the ability to simultaneously use a large numberof distinguishable probes. For example, when using emission of visiblelight as the detectable signal provided by the probe in response toexposure to radiation, multiple distinguishable probes, which eachcontain a single semiconductor nanocrystal of a respectively differentemission color (e.g., blue probe, green probe, red probe) may besimultaneously used. Moreover, a much greater number of distinguishableprobes may be simultaneously used when each probe contains a pluralityof semiconductor nanocrystals, all bound to a single three-dimensionallinking agent in the same probe (e.g., blue-green probe, green-redprobe, blue-red probe, blue-green-red probe). Still further increases incombinations of semiconductor nanocrystals bonded to a three-dimensionallinking agent structure can be achieved by varying the number ofidentically emitting semiconductor nanocrystals bonded to thethree-dimensional linking agent in the same probe in order to providedifferent intensities of detectable signals (e.g. adding a secondblue-emitting semiconductor nanocrystal to a blue-red probe to obtain ablue-blue-red probe, or adding another red-emitting semiconductornanocrystal to a blue-red probe to achieve a blue-red-red probe). Thisfurther increases the total number of probes which can be simultaneouslydistinguished. Similar benefits can be obtained when the detectablesignal or signals provided by the semiconductor nanocrystals in theprobe result from scattering (including diffraction) or absorptionresulting from exposure of the probe to radiation.

[0068] Similar to the incorporation of multiple semiconductornanocrystals in a single three-dimensionally structured linking agent,multiple affinity molecules may be linked to the same three-dimensionallinking agent structure to allow a plurality of detectable structures(including combinations of detectable structures) to be distinguishablyand simultaneously detected by each semiconductor nanocrystal probe.

[0069] In an illustration of the use of multiple affinity molecules ineach semiconductor nanocrystal probe in testing for Down's syndrome, asubset of the DNA sequences present on a particular chromosome in thehuman body, such as chromosome 21, may serve as the affinity moleculesof a semiconductor nanocrystal probe when attached, in the form of aplurality of separate single stranded DNA fragments, to athree-dimensionally structured linking agent linked to one or more redemitting nanocrystals. A subset of the DNA sequences present on adifferent chromosome, such as chromosome 3, may serve as the singlestranded DNA affinity molecules of another probe when similarly attachedto a different three-dimensionally structured linking agent linked toone or more green emitting nanocrystals. A material comprising a totalDNA sample from a human patient (or an amniocentesis sample), whereinare present one or more detectable substances in the form of singlestranded DNA, may be treated with these semiconductor nanocrystalprobes, resulting in the bonding of the single stranded DNA affinitymolecules of the probes with the single stranded DNA detectablesubstances. This bonding results in the formation of double stranded DNA(in one or both probes), indicative of the presence of one or more DNAsequences (i.e., DNA sequences represented by the single stranded DNAdetectable substances) in the DNA sample. This step may be followed witha step of detecting the bonding of the single stranded DNA affinitymolecules with the single stranded DNA detectable substances by, forexample, adding to the material, which contains the detectablesubstances and has been treated with the semiconductor nanocrystalprobes, a double stranded DNA-binding dye molecule (which may fluoresceblue). The amount of double stranded DNA-binding dye molecules present(determined by amount of blue fluorescence) on a semiconductornanocrystal probe, may be indicative of the amount of double strandedDNA associated with the semiconductor nanocrystal probe. Thus, the bluefluorescence from the probe containing DNA from chromosome 21 indicatesthe bonding of single stranded DNA affinity molecules from chromosome 21with complementary single stranded DNA detectable substances fromchromosome 21, to form double stranded DNA; and the blue fluorescencefrom the probe containing DNA from chromosome 3 indicates the bonding ofsingle stranded DNA affinity molecules from chromosome 3 withcomplementary single stranded DNA detectable substances from chromosome3, to form double stranded DNA.

[0070] In this test for Down's Syndrome, the semiconductor nanocrystalprobe comprising single stranded DNA affinity molecules from chromosome3, which emits green light, may serve as a reference probe, wherein theratio of emitted green light to emitted blue light represents thereference amount of double stranded DNA present on a semiconductornanocrystal probe. The semiconductor nanocrystal probe comprising singlestranded DNA affinity molecules from chromosome 21, which emits redlight, may serve as the test probe, wherein the ratio of emitted redlight to emitted blue light (from the test probe) may be compared to theratio of green light to blue light from the reference probe. Adifference between the test and reference ratios may indicate extra orfewer copies of the test chromosome (chromosome 21), in this caseindicating Down's Syndrome. The number of such tests which may besimultaneously performed may be significantly increased by the use of aplurality of colors in each of a plurality of semiconductor nanocrystalprobes.

[0071] As stated above, the three-dimensional linking agent structuremay comprise an organic or inorganic structure, and may be a porous ornon-porous solid, or hollow. When the three-dimensional linking agentstructure is a porous (or non-porous) solid the semiconductornanocrystal may be embedded therein, while the semiconductor nanocrystalmay be encapsulated in a hollow three-dimensional linking agentstructure. Whatever the choice of material, it will be appreciated thatwhenever the semiconductor nanocrystal is incorporated into the interiorof the three-dimensional structure of the linking agent, e.g., into a“polymer sphere”, the material comprising the linking agent must both(1) allow a first energy to be transferred from an energy source to theone or more semiconductor nanocrystals (exposing the semiconductornanocrystal to energy), and (2) allow a second energy, provided by theone or more semiconductor nanocrystals in response to exposure to thefirst energy, to be either detected or transferred to a proximalstructure. These transfers of energy may be accomplished by thethree-dimensional linking agent being transparent to the first and/orsecond energies, and/or by the three-dimensional linking agent beingcapable of converting the first and/or second energies to a form whichstill enables the semiconductor nanocrystal probe to either provide adetectable signal or transfer energy to a proximal structure in responseto exposure to energy.

[0072] When the three-dimensional linking agent comprises an organicmaterial, the organic material may comprise, for example, one or moreresins or polymers. The semiconductor nanocrystals may be linked to thethree-dimensional linking agent by physically mixing the semiconductornanocrystals with the resin(s) or polymer(s), or may be mixed with themonomer(s) prior to polymerization of the monomer(s) to form thepolymer(s). Alternatively, the semiconductor nanocrystals may be linkedto the three-dimensional linking agent by covalent bonding to either themonomer or the resin or polymer, or the semiconductor nanocrystals maybe linked to the three-dimensional linking agent by adsorption(adherence to the exterior) or absorption (embedded, at least partially,into the interior). Examples of polymers which could be used as organicthree-dimensional linking agents include polyvinyl acetate,styrene-butadiene copolymers, polyacrylates, and styrene-divinylbenzenecopolymers. More than one polymeric chain may be present in thethree-dimensional linking agent, and more than one type of polymer maybe used in the three-dimensional linking agent. The final product couldbe a solid structure, a hollow structure, or a semi-solid porousstructure.

[0073] When the three-dimensional linking agent structure comprises aninorganic material, a glass structure such as a glass sphere couldcomprise the transparent structure used to encapsulate one or moresemiconductor nanocrystals therein. The semiconductor nanocrystals couldbe mixed with particles of a low melting point glass, with the mixturethen heated to form the desired three-dimensional structure, e.g., asphere. Alternatively, a porous glass such as a porous silica glasscould be formed into a desired shape (or applied over a solid substrateas a porous coating), followed by incorporation of the semiconductornanocrystals into the pores of the linking agent structure. Thepreviously described glass-coated semiconductor nanocrystals could alsobe modified to provide the three-dimensional linking agent structure ofthis embodiment, for example by providing the glass coating over a coreof such semiconductor nanocrystals or by sintering into athree-dimensional mass a plurality of such glass coated semiconductornanocrystals comprising the same or different semiconductornanocrystals.

[0074] An additional increase in the number of three-dimensionalstructured probes which can be distinguishably used may arise fromplacing one or more identical semiconductor nanocrystals in one of aplurality of substructures of the three-dimensionally structured probe,and organizing the various substructures of the probe in such a mannerto allow a large number of uniquely identifiable probes to be formed.For example, in a single probe, the three-dimensional structured linkingagent may comprise a first semiconductor nanocrystal in a first polymercomprising a first substructure, and a second semiconductor nanocrystalin a second polymer immiscible with the first substructure comprisingsecond substructure.

[0075] One example of the arrangement of these substructures is a manneranalogous to the various layers of an onion. In such a construction,different arrangements of several differently emitting semiconductornanocrystals positioned in the various substructure layers may bedistinguished from one another. Therefore, a probe containing an innercore of blue semiconductor nanocrystals, encapsulated by a firstsubstructure layer of red semiconductor nanocrystals, which isencapsulated by a second substructure layer of green semiconductornanocrystals may be distinguished from a probe containing an inner coreof green semiconductor nanocrystals, encapsulated by a firstsubstructure layer of blue semiconductor nanocrystals, which isencapsulated by a second substructure layer of red semiconductornanocrystals. Thus, arranging the different substructures of thesemiconductor nanocrystal probe further increases the number ofdistinguishable probes which may be simultaneously used.

[0076] Additionally, various probes whose substructures are assembled indifferent arrangements may be distinguished. For example, a probe whichcomprises red, green and blue semiconductor nanocrystal substructuresordered in an onion-like arrangement may be distinguished from a probewhich comprises red, green, and blue semiconductor nanocrystalsubstructures ordered in a soccer ball-like arrangement.

[0077] Therefore, there are a number of different manipulations of thesemiconductor nanocrystals in the probe which results in a very largenumber of distinguishable probes. These manipulations include: varyingthe combinations of different semiconductor nanocrystals in the probe,varying the concentrations of similar and different semiconductornanocrystals in the probe, incorporating semiconductor nanocrystals intoa plurality of substructures in the probe, and varying the arrangementof such substructures containing semiconductor nanocrystals in theprobe.

[0078] The incorporation of multiple nanocrystals and/or multipleaffinity molecules into a single probe can be demonstrated in the use ofthe probes as the stationary phase in a screen for various nucleic acidsequences, where the nucleic acid sequences in the material beinganalyzed constitute the mobile phase.

[0079] A plurality of probes can be prepared which may each comprise aunique combination of semiconductor nanocrystals with similar or variedemission wavelengths. Associated with each probe having a uniquesemiconductor nanocrystal combination is a unique combination of one ormore affinity molecules comprising one or more known nucleic acidsequences. In this context, the term “nucleic acid sequence” should beunderstood to include single or double stranded ribonucleic acid (RNA)or deoxyribonucleic acid (DNA) molecules or chemical or isotopicderivatives thereof, each molecule comprising two or more nucleic acidmonomers. A plurality of unidentified nucleic acid sequences comprisingthe detectable substances in a mobile phase material being analyzed maynow be exposed to the above described plurality of probes, e.g. flowedover the stationary phase probes.

[0080] The detection, i.e., the identification of the nucleic acidsequences in the mobile phase bound to the probes involves two aspects.First of all the occurrence of a bonding event must be ascertained.Secondly the identification of which probe, and therefore which nucleicacid sequence or sequences (affinity molecules) of the probe, are boundto the nucleic acid sequences being analyzed. The bonding event itselfmay be determined by detection of a tag (e.g., a dye molecule) which hasbeen previously attached onto all of the nucleic acid sequences beinganalyzed. When bonding occurs, the presence of the tag will correspondspatially to a certain probe or probes. The identification of the typeof nucleic acid sequence or sequences may be determined by the detectionof the signal which corresponds to a unique combination of semiconductornanocrystals within the probe or probes involved in the bonding. Forexample, the probes and material being analyzed may be exposed toradiation of a type which will result in provision of detectable signalsfrom both the dye molecule and the particular probe or probes bonded tothe mobile phase nucleic acid sequences. A spatially identifiable groupof signals from both the dye molecules and semiconductor nanocrystalscan then be detected. The first signal, emanating from the nucleic acidsequences being identified, signifies the presence of a bonded nucleicacid sequence or sequences of any sequence type. The second detectablesignal, emanating from the probe (and the semiconductor nanocrystalstherein), identifies the type of nucleic acid sequence or sequenceswhich are bonded to the probe, by virtue of the known type of nucleicacid sequence or sequences forming the affinity molecule(s) of theprobe.

[0081] For example, the material being analyzed and the probes could beexposed to electromagnetic radiation from a laser light source of afrequency at which the dye is excitable and which will also excite thesemiconductor nanocrystals in the probe. The resulting detectablesignals from the dye molecules and the probe or probes, could be visiblelight emissions of one or more frequencies signifying the presence ofbonded nucleic acid sequences (the light from the dye molecules) and theidentity of the particular probe bonded thereto (the light from thesemiconductor nanocrystals in the probe). When the spatial locations ofboth the dye molecule emission and the probe emission correspond, thiswould signify the presence of particular nucleic acid sequences bondedto particular probes known to emit light of the detected frequencies.

[0082] Thus, once bonded to one or more nucleic acid sequences from themobile phase being analyzed, a plurality of similar or different probesmay then be identified according to the unique combination ofsemiconductor nanocrystals present in each probe. The probes may beidentified either one after the other or simultaneously. Theidentification of each probe then allows the identification of theunique nucleic acid sequence or combination of nucleic acid sequencesbound to the probe by way of the known nucleic acid sequence comprisingthe affinity molecule on the surface of each probe. In this way, a largenumber of different nucleic acid sequences may be rapidly screened andidentified.

[0083] It should be noted that while it is contemplated that eachaffinity molecule comprising a unique, known nucleic acid sequence orsequences will be specifically bonded to a particular unidentifiednucleic acid sequence or sequences being analyzed for, thus makingidentification precise, other uses may be contemplated. For example, aprobe could be designed having, as its affinity molecule portion, aparticular known nucleic acid sequence or sequences which would bebondable to an entire group of related unidentified nucleic acidsequences, thus permitting use of the probe as a broad identification orscreening agent.

f. Exposure of the Probe to Energy and Detection ofEmission/Absorption/Scattering

[0084] Upon exposure of the semiconductor nanocrystal probe to energy,some or all of the energy may be transferred to the semiconductornanocrystal probe. In response to exposure to this energy, thesemiconductor nanocrystal probe may provide a plurality of detectablesignals. These detectable signals include (1) emission ofelectromagnetic radiation, (2) absorption of radiation, and (3)scattering, including diffraction, of radiation.

[0085] The emission properties of the semiconductor nanocrystal probemay be very useful in a plurality of applications. As previouslymentioned, the semiconductor nanocrystal probe of the invention iscapable of being excited over a broad bandwidth, yet exhibits emissionin a narrow wavelength band, in contrast to the dye molecules used inthe prior art. Thus electromagnetic radiation of wavelength ranging fromx-ray to ultraviolet to visible to infrared waves may be used to excitethe semiconductor nanocrystals in the probe. In addition, thesemiconductor nanocrystals are capable of excitation from bombardmentwith a particle beam such as an electron beam (e-beam). Furthermore,because of the broad bandwidth at which the semiconductor nanocrystalsare excitable, one may use a common excitation source for thesimultaneous excitation of several probes, e.g., several probes whichgive off radiation at different frequencies, thus permittingsimultaneous excitation and detection of the presence of several probesindicating, for example, the presence of several detectable substancesin the material being examined.

[0086] Thus, for example, a laser radiation source of a given frequency,e.g., blue light, may be used to excite a first semiconductornanocrystal probe capable of emitting radiation of a second frequency,e.g., red light, indicating the presence, in the material beingilluminated, of a first detectable substance to which the particular redlight-emitting semiconductor nanocrystal probe has bonded. At the sametime, the same blue light laser source may also be exciting a secondsemiconductor nanocrystal probe (in the same material) capable ofemitting radiation of a third frequency, e.g., green light, indicatingthe presence, in the material being illuminated, of a second detectablesubstance to which the particular green light-emitting semiconductornanocrystal probe has bonded. Thus, unlike the prior art, multipleexcitation sources need not be used (because of the broad bandwidth inwhich the semiconductor nanocrystal probe of the invention is capable ofbeing excited), and the narrow band of emission of the specificsemiconductor nanocrystals in each probe makes possible the eliminationof sequencing and/or elaborate filtering to detect the emittedradiation.

[0087] Another detectable signal provided by a semiconductor nanocrystalprobe in response to radiation is absorption. The presence of thesemiconductor nanocrystal probe, bound to a detectable substance in abiological material, may be indicated by detection of absorption ofradiation by the semiconductor nanocrystal probe. Since thesemiconductor nanocrystal probe has such a wide wavelength band ofabsorption, detection of the semiconductor nanocrystal probe may becarried out over a wide range of wavelengths, according to therequirements of the detection process. For example, many biologicalmaterials strongly absorb visible and ultraviolet radiation, but do notstrongly absorb x-ray radiation. Such a biological material containing adetectable substance may be treated with a semiconductor nanocrystalprobe. Presence of the semiconductor nanocrystal probe bonded with thedetectable substance may then be indicated by detection of an absorptionof x-rays.

[0088] The semiconductor nanocrystal probe of the invention may alsoprovide a detectable scattering signal in response to exposure toenergy. This detectable scattering signal may be a diffraction signal.Thus, for example, presence of a detectable substance within a materialtreated with a semiconductor nanocrystal probe (wherein thesemiconductor nanocrystal probe is bonded to the detectable substance)may be indicated by the detection of a change in the scattering crosssection or in diffraction of radiation upon exposure of the material toradiation.

[0089] The semiconductor nanocrystal probe of the invention may also beused in such a way that, instead of providing a detectable signal inresponse to radiation, it transfers energy to a proximal structure. Thisproximal structure, in response to the energy transfer, may then (1)provide a detectable signal, (2) undergo chemical or conformationalchanges, (3) transfer energy to a second proximal structure, or (4) anycombination thereof. This may be achieved by introducing thesemiconductor nanocrystals and the material together by any of the abovemethods, and then exposing the material to energy. It should be notedthat a proximal source may be used to transfer energy from the proximalsource to the probe (as will be described below) in contrast to theaforesaid transfer of energy from the probe to a proximal structure.

g. General Use of the Probe

[0090] In general, the probe may be used in treating a material todetermine the presence of a detectable substance by introducing theprobe, for example, dispersed in a suitable carrier such as an aqueoussolution (e.g., an saline solution), into the material to permit theaffinity molecule of the probe to bond to the detectable substance (ifsuch detectable substance is present in the material). Afterintroduction of the probe into the material, unbonded probes may beoptionally removed from the material, leaving only bonded probes. Ineither event, the material (and probes therein) may be exposed to anenergy source capable of causing the probe(s) to provide a detectablesignal. When the unbonded probes have not been removed, presence of thebonded probes can be determined (and distinguished from the unbondedprobes) by a plurality of methods, including determining the spatialsegregation of more intense detectable signals arising as a result ofthe localization of the bonded probes, as opposed to random dispersion(resulting in spatially random detectable signals) of the unbondedsemiconductor nanocrystal probes.

[0091] As an alternative to adding the semiconductor nanocrystal probeto the material, the material may be in a carrier, such as an aqueoussolution, and this material may be introduced into a compartmentcontaining the semiconductor nanocrystal probe. The semiconductornanocrystal probe may itself be in a carrier, or may be attached to asolid support. Presence of the detectable substance within the materialmay be determined by any method which is capable of indicating thebonding of the affinity molecule of the probe to the detectablesubstance. This may be accomplished, for example, by separatingcomponents of the material and exposing the components of the materialto radiation, wherein a semiconductor nanocrystal probe, if present, mayprovide a detectable signal in response to exposure to radiation.

[0092] The carrier mentioned above is any type of matter that has littleor no reactivity with the semiconductor nanocrystal probe, and enablesstorage and application of the semiconductor nanocrystal probe to thematerial to be treated. Such a material will often be a liquid,including many types of aqueous solutions, including biologicallyderived aqueous solutions (e.g. plasma from blood). Other liquidsinclude alcohols, amines, and any other liquid which neither reacts withnor causes the dissociation of the components of the semiconductornanocrystal probe. The carrier also comprises a substance which will notinterfere with the treatment or analysis being carried out by the probein connection with the detectable substance in the material.

[0093] A further use of the semiconductor nanocrystal probe of theinvention is to provide a detectable signal in response to energytransferred from one or more spatially proximal sources. In thiscontext, “energy transfer” is meant the transfer of energy from oneatom, molecule, or any other substance (e.g. a polymer, a gel, a lipidbilayer, etc.) to another atom, molecule, or any other substance byeither (1) a radiative pathway (e.g., emission of radiation by a firstatom or molecule followed by scattering—including diffraction—and/orabsorption of the emitted radiation by a second atom or molecule); or(2) a non-radiative pathway (e.g., fluorescence resonance energytransfer, or FRET, from a first atom or molecule to a second atom ormolecule). By use of the term “proximal source” is meant an atom, amolecule, or any other substance which is capable of transferring energyto and/or receiving energy transferred from another atom or molecule orany other substance. By use of the term “spatially proximal source” ismeant a proximal source spaced sufficiently close to enable energy to betransferred from a proximal source to a semiconductor nanocrystal probe.For example, in the case of FRET, a spatially proximal source comprisesa proximal source spaced 10 nm or less from the semiconductornanocrystal probe. In the case of the transfer of radioactive energy, aspatially proximal source comprises a proximal source spaced 1 μm orless from the semiconductor nanocrystal probe.

[0094] The energy transferred from a proximal source to thesemiconductor nanocrystal probe may originate from the proximal source(e.g., radioactive decay of an atom or atoms within the proximal source)or may arise as a result of excitation by an energy source separate fromthe proximal source (e.g., excitation of a proximal source dye moleculeby a laser) as will be explained below. An illustration of a radiativepathway of energy transfer is the transfer of gamma radiation from aradioactive nucleus (of the proximal source) to a semiconductornanocrystal probe. The transferred gamma radiation may then be absorbedby the semiconductor nanocrystal probe, which, in response to absorptionof the gamma radiation, provides a detectable emission signal ofelectromagnetic radiation. An illustration of a non-radiative pathway isactivation of the semiconductor nanocrystal by a FRET from a proximalsource which has been externally excited, as will be described below.

[0095] Such a spatially proximal energy transfer may be useful inmeasuring the concentration of the proximal source, as well as thedistance of the proximal source from the probe. Spatially proximalenergy transfer can also be used in the detection of an event whichcauses the source from which energy is transferred to become spatiallyproximal to the probe.

[0096] One illustration of a spatially proximal energy transfer using asemiconductor nanocrystal probe is as a concentration indicator, whereinthe semiconductor nanocrystal probe, in essence, acts as an energytransfer reporter. That is, the semiconductor nanocrystal probe, forexample, may provide a detectable emission signal, the strength of whichis a function of the local concentration of proximal sources from whichthe energy is transferred. This permits the probe to be used todetermine the concentration of proximal sources from which energy istransferred. A possible application of this method would be to measurethe amount of a zinc finger protein, such as the RAG1 protein,synthesized by a cell during a specific length of time using apulse-chase experiment. The cell mixture may be pulsed with an additionof radioactive zinc ions to the growth medium and may, after a specificlength of time, be chased by addition of non-radioactive zinc ions inlarge excess (e.g., greater than 100-fold) of the radioactive zinc ions.Such a pulse-chase experiment will result in one or more radioactivezinc ions incorporated only in zinc containing proteins synthesizedduring the specified length of time between the pulse and the chase. Thecells may then be lysed to yield a soluble cell extract comprising oneor more zinc containing proteins. A semiconductor nanocrystal probecomprising an affinity molecule, such as an antibody, which selectivelybonds to a particular zinc finger protein may then be added to thesoluble cell extract, allowing the semiconductor nanocrystal probe tobond to the particular zinc finger protein. The concentration of theparticular zinc finger protein, comprising one or more radioactive zincions, and acting as the proximal source from which energy istransferred, bonded to semiconductor nanocrystal probe may be indicatedby a detectable signal provided by the semiconductor nanocrystal probein response to energy transferred from the radioactive zinc ion of thebonded particular zinc finger protein.

[0097] Another illustration of a spatially proximal energy transferusing the semiconductor nanocrystal probe is as a distance indicator.The strength of the detectable signal, for example, an emission, from asemiconductor nanocrystal probe is a function of the distance (providedthat the distance is less than about 1 μm) between the semiconductornanocrystal probe and the proximal source from which energy istransferred. Therefore, the detectable signal provided by thesemiconductor nanocrystal probe may serve as an indicator of thedistance between the semiconductor nanocrystal probe and the proximalsource from which energy is transferred. A possible application for thisis in the ability to determine spatial proximity of individual subunitsof a multi-subunit complex within a cell, such as a transcriptionalinitiation complex, a ribosome, a lipid-lipoprotein complex, etc. Forexample, a semiconductor nanocrystal probe may bond with a proteinsubunit of a ribosome, while a RNA subunit of the ribosome may belabeled with a radioactive phosphorous atom, which serves as theproximal source from which energy is transferred (in this illustration,the energy transferred from the proximal source to the semiconductornanocrystal probe originates from the proximal source). Since thestrength of the emission of a detectable signal is a function of thedistance between the semiconductor nanocrystal probe and the proximalsource from which energy is transferred, the signal provided by thesemiconductor nanocrystal probe bonded to the protein subunit indicatesthe approximate distance between the portion of the protein subunitbonded to the semiconductor nanocrystal probe and the portion of the RNAwhich contains the radioactive phosphorus atom from which the energy istransferred.

[0098] The spatially proximal energy transfer use of the semiconductornanocrystal probe also may be utilized to detect the occurrence of anevent. This event, for example, may cause the source from which energyis transferred to be located spatially proximal to the semiconductornanocrystal probe. Since the detectable signal is a function of thedistance between the proximal source from which energy is transferredand the semiconductor nanocrystal probe, the signal provided by thesemiconductor nanocrystal probe may yield information reflective of anevent which causes the source to be sufficiently proximal (less thanabout 10 nm) to enable energy to be transferred from the proximal sourceto the semiconductor nanocrystal probe. By way of illustration, asemiconductor nanocrystal probe may bond with a thyroid hormone receptormolecule. A thyroid hormone such as thyroxine may be labeled with aradioactive iodine atom, which serves as the source from which energy istransferred. An event which causes the thyroxine to bond to the thyroidhormone receptor will also cause the radioactive iodine atom in thethyroxine to be spatially proximal to the semiconductor nanocrystalprobe. Therefore, this bonding event will cause energy to be transferredfrom the radioactive iodine atom to the semiconductor nanocrystal probewhich may provide a detectable signal in response to the energytransfer. The detectable response will thus serve as an indicator of theevent of thyroxine bonding to the thyroid hormone receptor.

[0099] The energy transferred from one or more proximal sources to oneor more semiconductor nanocrystal probes may either originate from theproximal source (as in the example of radioactive decay of an atom oratoms within the proximal source), or may arise as a result ofexcitation of the one or more proximal sources by an energy sourceseparate from the proximal sources. By use of the term “energy sourceseparate from the proximal source” is meant any source of radiation orany other energy which transfers energy to the proximal source. Theenergy source separate from the one or more proximal sources may eitherbe spatially distant or spatially proximal to the proximal source fromwhich energy is transferred to the semiconductor nanocrystal probe.Thus, the energy may be transferred from a spatially distant energysource such as, for example, a laser or particle beam; or the energy maybe transferred from a second spatially proximal source from which secondproximal source the energy transferred may either originate, or arise asa result of excitation by an energy source separate from the secondproximal source. For example, a laser beam may be used to excite asecond proximal source, the second proximal source then excites thefirst proximal source, and the first proximal source excites thesemiconductor nanocrystal probe; or a second proximal source may be aradioactive atom which excites the first proximal source which excitesthe semiconductor nanocrystal probe. It will be understood that morethan two proximal energy sources can be utilized to transfer energy in acascading effect. Included in pathways of excitation of the proximalsource by a separate source is the case where the separate source is aparticle beam which, when the proximal source is exposed to the particlebeam, may cause a nuclear event in the proximal source. The proximalsource may then transfer energy to the semiconductor nanocrystal probeas a result of the nuclear event caused by exposure of the proximalsource to the particle beam.

[0100] When the excitation of the proximal source arises as a result ofenergy transferred from a separate energy source (e.g., a laser beam)the energy transfer from the proximal source to the semiconductornanocrystal probe may be accomplished by FRET, as previously mentioned.Thus, an energy source separate from the proximal source, such as alaser, may excite a proximal source. The proximal source, as a result ofrelaxing from an excited state, may transfer energy via fluorescenceresonance energy transfer to the semiconductor nanocrystal probe whenthe proximal source is less than about 10 nm from the semiconductornanocrystal probe. The semiconductor nanocrystal probe may then providea detectable signal such as electromagnetic radiation in response to theenergy transfer from the proximal molecule. An illustration of both theexcitation of the proximal molecule by an energy source separate fromthe proximal energy source and the use of FRET as the pathway of energytransfer from the proximal source to the probe may be derived from thepreviously described ribosomal example. In contrast to the previousexample which used an RNA subunit of the ribosome labeled with aradioactive phosphorus atom as the proximal source, a dye molecule maybe attached to the RNA subunit instead of the radioactive phosphorousatom. The proximal source RNA subunit with attached dye molecule maythen be excited by a separate source, for example a laser beam. Theexcited proximal source RNA subunit may transfer energy to asemiconductor nanocrystal probe by way of a non-radiative energytransfer pathway such as FRET, which may provide a detectable signal inresponse to the energy transferred from the proximal source RNA subunit.

[0101] The use of a proximal source to transfer energy to asemiconductor nanocrystal probe may be modified in such a way as toenable a proximal source to transfer energy to a plurality ofsemiconductor nanocrystal probes. By way of illustration, in theprevious example using an RNA molecule labeled with a dye molecule asthe proximal molecule, a plurality of RNA proteins may be labeled, eachwith a differently emitting semiconductor nanocrystal probe.Fluorescence resonance energy may be transferred from the dye moleculeto one or more of the differently emitting semiconductor nanocrystalprobes. The detectable signals provided by the one or more differentlyemitting semiconductor nanocrystal probes may then signify proximitybetween the dye and the one or more semiconductor nanocrystal probes.

[0102] Since semiconductor nanocrystals of specific wavelength emissionmay be selected for use in a particular probe, a semiconductornanocrystal probe may be exposed to, for example, a radioactive atomemitting gamma radiation from a proximal source, and the wavelength ofthe emission from the semiconductor nanocrystal probe, in response toexposure to gamma radiation from the proximal source, may be selected tobe ultraviolet radiation, according to the nature of the semiconductornanocrystal within the semiconductor nanocrystal probe. Alternatively,the wavelength of the emission of the semiconductor nanocrystal inresponse to exposure to, for example, gamma radiation from the proximalsource may be selected to be red light. The ability to provide multipleand selectable different emissions in response to exposure to theidentical radiation allows a plurality of differently emittingsemiconductor nanocrystal probes to be used simultaneously. Thesimultaneous use of a plurality of probes which each emit differentwavelengths of electromagnetic radiation can be used, for example, in aconfiguration where proximity between a specific semiconductornanocrystal probe and a source from which energy is transferred to thesemiconductor nanocrystal probe may be determined by the specificwavelength of the emission from the semiconductor nanocrystal probe. Forexample, three semiconductor nanocrystal probes which differ in thevisible light they emit (e.g., blue, green, and red emittingsemiconductor nanocrystal probes) could be attached to portions of anassociation of molecules (e.g., an organelle). Presence of a certainmolecule with a radioactive atom attached (therefore acting as theproximal source) in proximity to one specific semiconductor nanocrystalprobe results in emission of a specific color, indicating proximitybetween the certain molecule and the specific semiconductor nanocrystalprobe and its associated affinity molecule.

[0103] Similar to the use of multiple semiconductor nanocrystals, it ispossible to use multiple proximal sources capable of transferring energyto one or more semiconductor nanocrystal probes.

[0104] Similar to the process in which energy is transferred from one ormore proximal sources to one or more semiconductor nanocrystal probes,energy may also be transferred from one or more semiconductornanocrystal probes to one or more proximal structures in response toexposure of the semiconductor nanocrystal probe to energy. The term“proximal structure” as used herein may be an atom, a molecule, or anyother substance (e.g. a polymer, a gel, a lipid bilayer, and anysubstance bonded directly to a semiconductor nanocrystal probe) which iscapable of receiving energy transferred from another atom or molecule orother substance (including a semiconductor nanocrystal probe). Theproximal structure, in response to the energy transferred from thesemiconductor nanocrystal probe, may (1) provide a detectable signal,(2) undergo chemical and/or conformational changes, (3) transfer energyto one or more second proximal structures, or (4) any combinationthereof. As used herein, a “second proximal structure” is a proximalstructure to which energy is transferred from a first proximal structurewhich has received energy from a semiconductor nanocrystal probe. Thesecond proximal structure, in response to the energy transferred fromthe first proximal structure may (1) provide a detectable signal, (2)undergo chemical and/or conformational changes, (3) transfer energy toone or more third proximal structures (where a “third proximalstructure” is one to which energy has been transferred from a secondproximal structure), or (4) any combination thereof. It will beunderstood that the transfer of energy between proximal structures maybe further extended beyond a third proximal structure in a cascadingeffect.

[0105] An illustration of the use of a semiconductor nanocrystal probeto transfer energy to a proximal structure which provides a detectablesignal is as follows. A semiconductor nanocrystal probe may be used toprovide an emission of a narrow wavelength band in the blue region ofvisible light in response to excitation over a broad wavelength band ofradiation.

[0106] When this semiconductor nanocrystal probe is spatially proximalto a dye molecule (the dye molecule herein is acting as the proximalstructure), the dye molecule may then become excited upon transfer ofenergy from the semiconductor nanocrystal probe. The excited dyemolecule may then be capable of providing a detectable red lightemission in response to excitation by the energy transfer from thesemiconductor nanocrystal.

[0107] An illustration of the use of a semiconductor nanocrystal probeto transfer energy to a proximal structure which, in response to theenergy transferred from the semiconductor nanocrystal probe, undergoeschemical changes, is the use of semiconductor nanocrystals to breakcovalent bonds. A semiconductor nanocrystal probe may be exposed toenergy, and may then transfer energy to a proximal structure in responseto the exposure to energy. The energy transferred may be, for example,electromagnetic radiation which is capable of inducing a photolyticcleavage (or photolysis) of a covalent bond in a proximal structure.This action of photolysis may also result in the detachment of a portionof the proximal structure. This detached portion of the proximalstructure may be, for example, a molecule used for therapeutic purposessuch as a molecule with cytotoxic properties. This use of thesemiconductor nanocrystal probe to break covalent bonds may becontrolled in a dosage specific manner, according to the extent ofexposure of the semiconductor nanocrystal probe to radiation. Thiscontrol of the exposure of the semiconductor nanocrystal probe toradiation may result in control of the energy transferred to theproximal structure, which controls the photolytic cleavage of thecovalent bond, and ultimately controls the detachment of the portion ofthe proximal structure. Additionally, the portion of the proximalstructure may be detached in a spatially specific manner, according tothe specificity of the one or more affinity molecules of thesemiconductor nanocrystal probe.

[0108] This use of the semiconductor nanocrystal probe to break covalentbonds in the proximal structure may be particularly effective when theenergy transferred to the semiconductor nanocrystal probe has a longwavelength which is transparent to the material surrounding thesemiconductor nanocrystal probe. For example, a semiconductornanocrystal probe may be exposed to electromagnetic radiation from alaser which emits at a wavelength of 700 nm (infrared radiation).Materials such as biological materials absorb very little radiation at700 nm, but a semiconductor nanocrystal probe may absorb radiation at700 nm. It is common for photolytic cleavages to require ultravioletradiation for activation. An advantage of the semiconductor nanocrystalprobe of the invention is that it may be made to transfer energycorresponding to ultraviolet radiation when exposed to infraredradiation as a result of a process termed two-photon absorption.Two-photon absorption may occur when a semiconductor nanocrystal probeis exposed to radiation in such a way that it simultaneously absorbs twoquanta of radiation (i.e., two photons), and the resultant level ofexcitation of the semiconductor nanocrystal probe is twice as large asthe level of excitation the semiconductor nanocrystal probe would haveif it had absorbed a single quantum of radiation. By the physicalrelationship between energy and wavelength of radiation (E=hc/λ, where Eis energy, h and c are constants, and λ is wavelength), a level ofexcitation, corresponding to two quanta of a first type of radiationwith a certain wavelength, would correspond to the level of excitationcaused by absorption of a single quantum of a second type of radiationwith a wavelength half that of the first type of radiation. Thus, if asemiconductor nanocrystal probe simultaneously absorbs two photons withwavelength of 700 nm, the excitation level of the semiconductornanocrystal probe will be the same as the excitation level of asemiconductor nanocrystal probe which absorbs a single photon with awavelength of about 350 nm (ultraviolet radiation). A semiconductornanocrystal probe which has been excited by two-photon absorption maythus transfer energy, for example, by emitting electromagnetic radiationwith a shorter wavelength than the wavelength of the radiation to whichthe semiconductor nanocrystal probe was exposed.

[0109] As an illustration of the use of this two-photon absorption, asemiconductor nanocrystal probe, comprising one or more affinitymolecules which may specifically bond to one or more detectablesubstances representative of the presence of a cancerous cell or tissue,may be exposed to radiation from an infrared laser emitting at 700 nm.This semiconductor nanocrystal probe may then be excited by the infraredradiation (through the process of two-photon absorption), and may thenemit ultraviolet radiation (which has a shorter wavelength—e.g. about350 nm). This emitted radiation in the ultraviolet range (or energytransferred by some other process, such as by FRET) may then cause aphotolytic cleavage in a proximal structure, which results in acytotoxic molecule being detached from the proximal structure and actingas a toxin to the cancerous cell or tissue.

[0110] Another illustration of the response to the energy transferredfrom the semiconductor nanocrystal probe to the proximal structureresulting in the proximal structure undergoing chemical orconformational changes may result when the energy transferred from thesemiconductor nanocrystal probe to the proximal structure is heatenergy. This transfer of heat energy may result in a conformationalchange such as the heat-induced denaturation of a protein. Asemiconductor nanocrystal probe may be able to absorb radiation which isnot absorbed by the material surrounding the semiconductor nanocrystalprobe. In response to exposure of the semiconductor nanocrystal probe toradiation, the semiconductor nanocrystal probe may transfer heat energyto a proximal structure, resulting in a local heating of structuresproximal to the semiconductor nanocrystal probe. In response to thislocal heating, the proximal structure may (1) undergo a chemical orconformational change, and/or (2) transfer energy to a second proximalstructure. Thus, exposure of a material to radiation (to which radiationthe material is transparent) may result in local heating within thematerial. The heat energy transferred from the semiconductor nanocrystalto the proximal structure may then result in chemical or conformationalchanges in the proximal structure, and/or some or all of the heat energymay be transferred to a second proximal structure which itself couldundergo chemical or conformational changes and/or transfer some or allof the heat energy to a third proximal structure, and so on. As in theexample of the photolytically detached cytotoxic molecule, use of thesemiconductor nanocrystal probe to cause transfer of heat energy may becontrolled in a dosage specific manner, according to the extent ofexposure of the semiconductor nanocrystal probe to radiation.Additionally, the heat energy may be transferred in a spatially specificmanner, according to the specificity of the one or more affinitymolecules of the semiconductor nanocrystal probe.

[0111] The amount of heat energy transferred to a proximal structurefrom a semiconductor nanocrystal probe in response to exposure toradiation may be enough to generate a large amount of local heating dueto the high degree of stability and the large extinction coefficientscharacteristic of nanocrystals. In a specific example of the extent oflocal heating which may occur, when semiconductor nanocrystals (whichemit infrared radiation) are present in a tissue at a concentration ofabout 0.0001 grams of semiconductor nanocrystals per gram of tissue, andthese nanocrystals are exposed to an ultraviolet excitation source (or atwo photon absorption source capable of exciting with an ultravioletexcitation energy), the heat energy transferred by these semiconductornanocrystals over 1,000,000 photocycles (about one second of exposure toa saturating laser) in response to exposure to radiation may cause thetissue to increase in temperature by about 25° C. This large amount oflocal heating may be, for example, great enough to kill local cells andtissue; and therefore this use of the semiconductor nanocrystal probe totransfer heat energy may be applied to the treatment of cancerous cellsor other nefarious cells and tissues.

[0112] Energy transfer from one or more semiconductor nanocrystal probesto one or more proximal structures may take place in a manner similar toany of the previously described transfers of energy from one or moreproximal sources to one or more semiconductor nanocrystal probes.Therefore, a semiconductor nanocrystal probe may transfer energy to aproximal structure by way of radiative or non-radiative (e.g., FRET)pathways. The energy transferred from a semiconductor nanocrystal probeto a proximal structure by a radiative pathway may include particle andelectromagnetic radiation. The energy transfer from a semiconductornanocrystal probe to a proximal structure may occur as a result ofenergy transferred from an energy source separate from the semiconductornanocrystal probe. This energy source separate from the semiconductornanocrystal probe may either be a spatially distant energy source suchas, for example, a laser or particle beam; or the energy may betransferred from a spatially proximal source, as previously discussed.This includes, for example, a spatially distant energy source which maytransfer energy to a spatially proximal source, which may transferenergy to a semiconductor nanocrystal probe, which may transfer energyto a proximal structure.

[0113] Prior to using a semiconductor nanocrystal probe in a processcomprising exposure of the semiconductor nanocrystal probe to energy,the semiconductor nanocrystal probe may be used as a precursor which maybe subjected to further synthetic steps. These further synthetic stepsmay result in formation of a modified semiconductor nanocrystal probewhich has a different affinity molecule than the affinity molecule ofthe precursor semiconductor nanocrystal probe. For example, asemiconductor nanocrystal probe, having one or more nucleic acidmonomers as its affinity molecule portion, may serve as a precursor(primer) in a process for synthesizing DNA in large amounts, such aspolymerase chain reaction (PCR); and the final PCR product may be amodified semiconductor nanocrystal probe having an affinity moleculewith a greater number of nucleic acid monomers than the affinitymolecule of the precursor semiconductor nanocrystal probe. The syntheticsteps to which the semiconductor nanocrystal probe may be subjectedinclude, for example, any method of nucleic acid synthesis (by use ofthe term, “nucleic acid synthesis” it is meant any enzymatic process ofsynthesizing nucleic acid strands using nucleic acid monomers). In anysuch nucleic acid synthesis (including the above PCR case), theprecursor semiconductor nanocrystal probe is understood to comprise oneor more nucleic acid strands, each comprising a number of nucleic acidmonomers sufficient to allow the precursor semiconductor nanocrystal tobe used as a primer in a nucleic acid synthesis reaction such as PCR(the nucleic acid strands often having from 1 to about 50 nucleic acidmonomers) as the one or more affinity molecules portion of thesemiconductor nanocrystal probe. The term “nucleic acid strand” shouldbe understood to include a plurality of single or double strandedribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecules orchemical or isotopic derivatives thereof, each molecule comprising twoor more nucleic acid monomers. This nucleic acid strand affinitymolecule portion may be modified by extending the nucleic acid strandsby addition of nucleic acid monomers according to the desired sequenceof the nucleic acid synthesis (chains may vary in length from 1 morenucleic acid monomer than the precursor, or primer, to as much as500,000 nucleic acid monomers, or more if desired). This modifiedsemiconductor nanocrystal probe is understood to have all of theproperties and potential uses of any semiconductor nanocrystal probe.That is, the modified semiconductor nanocrystal probe is capable ofbonding with one or more detectable substances, and is capable ofproviding a detectable signal in response to exposure to energy. Thismay include, for example, use of the modified semiconductor nanocrystalprobe (comprising an affinity molecule with a modified DNA sequence) asa fluorescent marker in a plurality of nucleic acid based assays,including DNA sequencing assays and hybridization assays such asfluorescence in-situ hybridization and comparative genomichybridization.

[0114] Another advantage of the semiconductor nanocrystal probe (or asemiconductor nanocrystal compound) of the invention is in any processwhich involves elevated temperatures. As used herein, “elevatedtemperatures” are understood to include temperatures from roomtemperature (about 25° C.) up to the temperature at which the particularsemiconductor nanocrystal probe undergoes thermal degradation. Typicallythis may occur at temperatures of about 150° C. or even as low as 100°C. Because of the high degree of thermal stability of the semiconductornanocrystals, semiconductor nanocrystal probes (or semiconductornanocrystal compounds) may withstand use at elevated temperatures,including use in processes which comprise thermal cycling steps (i.e.,processes which comprise one or more steps in which the temperature iscycled between a low temperature and a high temperature, such as theaforementioned PCR). For example, as discussed above, a precursorsemiconductor nanocrystal probe may be used in PCR, which requiresmultiple steps in which the temperature is cycled between a lowtemperature (the DNA synthesis step) and a high temperature (the DNAstrand separation step). The high temperature of the PCR reactionmixture may be about 95° C., a temperature at which many dye moleculesdegrade. The thermal stability properties of the semiconductornanocrystal probe enable it to withstand the thermal cycling of PCR.

[0115] In addition to the use of semiconductor nanocrystal probes inPCR, the advantage of the high degree of thermal stability of thesemiconductor nanocrystal probes may be applied to any other processeswhich may require elevated temperatures, such as use in heat shockmethods, or methods using thermostable organisms or biomolecules derivedfrom thermostable organisms.

[0116] An illustration of the simultaneous use of a plurality ofdifferent semiconductor nanocrystal probes is when a plurality ofsemiconductor nanocrystal probes are used in flow cytometry analysis.Flow cytometry, as used in the prior art, involves contacting amaterial, containing cells, with one or more dyes, or dye conjugatedaffinity molecules, which are capable of detecting certain molecules orsubstances on the surface or interior of those cells. The presence ofthe dye molecules on the surface or interior of a cell (and, hence, thepresence of the certain molecule with which the dye interacts) isdetected by flowing the material through a compartment which istransparent to both the energy to which the material is exposed, and tothe detectable signal provided by the dye in response to exposure toenergy. As the cells are within the transparent compartment, the cellsare exposed to energy, such as electromagnetic radiation, which iscapable of being absorbed by the dye. The dye, as a result of exposureto the electromagnetic radiation, emits a detectable signal, such aselectromagnetic radiation of a different wavelength than that to whichthe material is exposed. When a plurality of dyes are used to indicatethe presence of a plurality of substances on the surface or interior ofthe cells, the material containing the cells may be flowed through aplurality of transparent compartments, and the presence of a pluralityof different dyes may be tested one at a time (i.e. consecutively) or afew at a time (maximum of three simultaneous detections).

[0117] In accordance with the invention, instead of using a dyemolecule, a material containing cells may alternatively be contactedwith a semiconductor nanocrystal probe (actually a plurality of probe,but all providing the same detectable signal in response to energy). Thesemiconductor nanocrystal probe may bond to one or more detectablesubstances, if any are present, on the surface or interior of the cells,to which the affinity molecules of the semiconductor nanocrystal probeare capable of bonding. Detection of the presence of the semiconductornanocrystal probe (and hence, the presence of one or more specificdetectable substances to which the semiconductor nanocrystal probe isbonded) may take place by first contacting the material containing thecells with the semiconductor nanocrystal probe. The material is thenflowed through a transparent compartment wherein the material is exposedto energy such as, for example, ultraviolet laser radiation. Thepresence of the semiconductor nanocrystal probe may be indicated by adetectable signal such as, for example, emission of red light, providedby the semiconductor nanocrystal probe in response to exposure toenergy. Detection of the detectable signal provided by the semiconductornanocrystal probe, therefore, may indicate the presence of one or moredetectable substances, on the surface or interior of cells, to which thesemiconductor nanocrystal probe is bonded.

[0118] Use of a plurality of groups of semiconductor nanocrystal probes(each of which groups provide the same detectable signal in response toexposure to energy) may be conducted in a manner similar to the aboveuse of a single semiconductor nanocrystal probe. The material containingthe cells may be contacted with a plurality of semiconductor nanocrystalprobes, and the material is then flowed through a plurality oftransparent compartments. In each compartment, the presence of aspecific semiconductor nanocrystal probe bonded to one or moredetectable substances may be indicated by a particular detectable signalprovided by the specific semiconductor nanocrystal probe. However,unlike the prior art, since each separate semiconductor nanocrystalprobe is capable of producing a detectable signal (in response toenergy) which is distinguishable from the detectable signals produced byother semiconductor nanocrystal probes which have been exposed to thesame energy, the presence of more than one semiconductor nanocrystalprobe, each bonded to one or more different detectable substances, maybe simultaneously detected in a single compartment.

[0119] Furthermore, methods of using one or more semiconductornanocrystal probes to detect one or more detectable substances on thesurface or interior of cells may not require flowing the materialthrough a transparent compartment, thereby extending the use of thesemiconductor nanocrystal probes to any cytometric method (i.e. anymethod which is used to detect the presence of detectable substances onthe surface or interior of cells). Instead of flowing thecell-containing material through a transparent compartment, the presenceof one or more of a plurality of semiconductor nanocrystal probes bondedto the cells may be detected by any technique capable of detecting thesignals from the different semiconductor nanocrystal probes in aspatially sensitive manner. Such spatially sensitive detection methodsinclude, for example, confocal microscopy and electron microscopy, aswell as the aforementioned flow cytometry.

[0120] The following examples will serve to further illustrate theformation of the semiconductor nanocrystal probes of the invention, aswell as their use in detecting the presence of a detectable substance ina material such as a biological material.

EXAMPLE 1

[0121] To illustrate the formation of the semiconductor nanocrystalcompound (comprising the semiconductor nanocrystals linked to a linkingagent) 20 ml. of a 5 mM solution of (4-mercapto)benzoic acid wasprepared with a pH of 10 using (CH₃)₄NOH.5H₂O. 20 mg oftris-octylphosphine oxide coated CdSe/CdS core/shell nanocrystals wereadded to the solution and stirred until completely dissolved. Theresultant nanocrystal/linking agent solution was heated for 5 hours at50-60° C. and then concentrated to a few ml by evaporation. Then anequal volume of acetone was added and the nanocrystals precipitated outof solution homogeneously. The precipitate was then washed with acetone,dried, and then can be stored.

[0122] The semiconductor nanocrystal compound prepared above can belinked with an appropriate affinity molecule to form the semiconductornanocrystal probe of the invention to treat a biological material todetermine the presence or absence of a detectable substance. That is,the semiconductor nanocrystal compound prepared above can be linked, forexample, with avidin or streptavidin (as the affinity molecule) to forman semiconductor nanocrystal probe to treat a biological material toascertain the presence of biotin; or the semiconductor nanocrystalcompound prepared above can be linked with anti-digoxiginen to form ansemiconductor nanocrystal probe to treat a biological material toascertain the presence of digoxiginen.

EXAMPLE 2

[0123] To illustrate the formation of a semiconductor nanocrystalcompound (comprising silica coated semiconductor nanocrystals linked toa linking agent) 200 μl of 3-(mercaptopropyl)-trimethoxysilane and 40 μlof 3-(aminopropyl)-trimethoxysilane were added to 120 ml of anhydrous25% (v/v) dimethylsulfoxide in methanol. The pH of this solution wasadjusted to 10 using 350 μl of a 25% (w/w) solution of (CH₃)₄)NOH inmethanol. 10 mg of CdS or ZnS or ZnS/CdS coated CdSe nanocrystals weredissolved into this solution (prepared, in the case of CdS, by atechnique such as the technique described in the aforementioned Peng,Schlamp, Kadavanich, and Alivisatos article; or in the case of ZdS, bythe technique described by Dabbousi et al. in “(CdSe)ZnS Core-ShellQuantum Dots: Synthesis and Characterization of a Size Series of HighlyLuminescent Nanocrystals,” Journal of Physical Chemistry B 101 pp9463-9475, 1997), stirred to equilibrate for several hours, diluted with200 ml of methanol with 150 μl of a 25% (w/w) solution of (CH₃)₄NOH inmethanol, then heated to boiling for minutes. This solution was thencooled and mixed with a 200 ml solution of 90% (v/v) methanol, 10% (v/v)water, containing 1.0 ml of 3-(trihydroxysilyl)propyl methylphosphonate,monosodium salt (42% w/w solution in water) and 40 μl of3-(aminopropyl)trimethoxysilane. This solution was stirred for twohours, then heated to boiling for fewer than five minutes, then cooled.Once cool, a solution of 4 ml of chlorotrimethylsilane in 36 mlmethanol, the pH of which had been adjusted to 10 using solid(CH₃)₄NOH.5H₂O, was mixed with the solution and stirred for one hour.This solution was then heated to boiling for 30 minutes, cooled to roomtemperature and stirred for several hours more. The solvent wasevacuated partially in vacuo at 60° C. This solution can be precipitatedto an oily solid with acetone. The semiconductor nanocrystal compoundmay then be redissolved in water, and in a variety of buffer solutionsto prepare it for linking it to an affinity molecule to form thesemiconductor nanocrystal probe of the invention to treat a biologicalmaterial to determine the presence or absence of a detectable substance.

[0124] Thus, the invention provides an semiconductor nanocrystal probecontaining a semiconductor nanocrystal capable, upon excitation byeither electromagnetic radiation (of either narrow or broad bandwidth)or particle beam, of emitting electromagnetic radiation in a narrowwavelength band and/or absorbing energy and/or scattering or diffractingsaid excitation, thus permitting the simultaneous usage of a number ofsuch probes emitting different wavelengths of electromagnetic radiationto thereby permit simultaneous detection of the presence of a number ofdetectable substances in a given material. The probe material is stablein the presence of light or oxygen, capable of being excited by energyover a wide spectrum, and has a narrow band of emission, resulting in animproved material and process for the simultaneous and/or sequentialdetection of a number of detectable substances in a material such as abiological material.

Having thus described the invention what is claimed is:
 1. Asemiconductor nanocrystal compound capable of linking to one or moreaffinity molecules and capable of, in response to exposure to a firstenergy, providing a second energy, said semiconductor nanocrystalcompound comprising: a) one or more semiconductor nanocrystals, eachcapable of, in response to exposure to said first energy, providing saidsecond energy; and b) one or more linking agents, at least a portion ofwhich said linking agents are linked to said one or more semiconductornanocrystals.
 2. The semiconductor nanocrystal compound of claim 1wherein said one or more semiconductor nanocrystals in said compound arecapable of receiving said first energy by fluorescence resonance energytransfer (FRET).
 3. The semiconductor nanocrystal compound of claim 1wherein said one or more semiconductor nanocrystals in said compound arecapable of providing said second energy by fluorescence resonance energytransfer (FRET).
 4. The semiconductor nanocrystal compound of claim 1wherein said one or more semiconductor nanocrystals in said compound arecapable of receiving said first energy by exposure to radiation.
 5. Thesemiconductor nanocrystal compound of claim 4 wherein each of said oneor more semiconductor nanocrystals is capable of absorbing saidradiation over a wide bandwidth.
 6. The semiconductor nanocrystalcompound of claim 1 wherein said second energy results from diffractionand/or scattering of said first energy by at least one of said one ormore semiconductor nanocrystals.
 7. The semiconductor nanocrystalcompound of claim 1 wherein said second energy results from absorptionof said first energy by at least one of said one or more semiconductornanocrystals.
 8. The semiconductor nanocrystal compound of claim 1wherein said one or more semiconductor nanocrystals in said compound arecapable of providing said second energy as electromagnetic radiationemitted by said semiconductor nanocrystals.
 9. The semiconductornanocrystal compound of claim 1 wherein each of said one or more linkingagents is capable of linking to said one or more affinity molecules. 10.The semiconductor nanocrystal compound of claim 1 wherein said one ormore linking agents include a glass coating on said one or moresemiconductor nanocrystals, and said glass coating is capable of beinglinked to said one or more affinity molecules.
 11. The semiconductornanocrystal compound of claim 10 wherein said glass coating on said oneor more semiconductor nanocrystals comprises a coating of silica glass.12. The semiconductor nanocrystal compound of claim 1 wherein at leastone of said one or more linking agents comprises: a) a first linkingagent linked to at least one of said one or more semiconductornanocrystals; and b) a second linking agent: i) linked to said firstlinking agent on said one or more semiconductor nanocrystals; and ii)capable of linking to said one or more affinity molecules.
 13. Thesemiconductor nanocrystal compound of claim 1 comprising two or moresemiconductor nanocrystals wherein, in response to exposure to saidfirst energy, said second energy provided by a first of said two or moresemiconductor nanocrystals is different than said second energy providedby a second of said two or more semiconductor nanocrystals.
 14. Asemiconductor nanocrystal compound capable of linking to one or moreaffinity molecules and capable of, in response to exposure to a firstenergy, providing a second energy, said semiconductor nanocrystalcompound comprising: a) one or more semiconductor nanocrystals, eachcapable of, in response to exposure to said first energy, providing saidsecond energy; and b) one or more first linking agents to which said oneor more semiconductor nanocrystals are linked, each of said one or morefirst linking agents capable of linking to: i) one or more secondlinking agents; or ii) one or more affinity molecules.
 15. Thesemiconductor nanocrystal compound of claim 14 wherein at least one ofsaid one or more first linking agents comprises a three-dimensionalshaped structure capable of having linked thereto said one or moresemiconductor nanocrystals.
 16. The semiconductor nanocrystal compoundof claim 15 wherein said three-dimensional shaped structure is capableof being linked, by embedding, to said one or more semiconductornanocrystals.
 17. The semiconductor nanocrystal compound of claim 15wherein said three-dimensional shaped structure is capable of beinglinked, by adherence, to said one or more semiconductor nanocrystals.18. The semiconductor nanocrystal compound of claim 15 wherein saidthree-dimensional shaped structure further comprises one or more organicmaterials.
 19. The semiconductor nanocrystal compound of claim 15wherein said three-dimensional shaped structure further comprises one ormore inorganic materials.
 20. The semiconductor nanocrystal compound ofclaim 15 wherein said three-dimensional shaped structure comprises aporous solid structure which encapsulates said one or more semiconductornanocrystals.
 21. The semiconductor nanocrystal compound of claim 15wherein said three-dimensional shaped structure comprises a non-poroussolid structure which encapsulates said one or more semiconductornanocrystals.
 22. The semiconductor nanocrystal compound of claim 15wherein said three-dimensional shaped structure comprises a hollowstructure which encapsulates said one or more semiconductornanocrystals.
 23. The semiconductor nanocrystal compound of claim 15wherein said three-dimensional shaped structure comprises a layeredstructure having two or more layers.
 24. The semiconductor nanocrystalcompound of claim 15 wherein said three-dimensional shaped structurecomprises a medium transparent to: i) said first energy to which saidone or more semiconductor nanocrystals is exposed; and ii) said secondenergy provided by said semiconductor nanocrystals in response to saidexposure to said first energy.
 25. The semiconductor nanocrystalcompound of claim 15 wherein said three-dimensional shaped structurecomprises a medium: i) capable of transferring said first energy to saidone or more semiconductor nanocrystals; and ii) transparent to saidsecond energy provided by said semiconductor nanocrystals in response tosaid exposure to said first energy.
 26. A semiconductor nanocrystalcompound capable of linking to one or more affinity molecules andcapable of emitting electromagnetic radiation in a narrow wavelengthband when exposed to radiation comprising: a) one or more semiconductornanocrystals, each capable of emitting electromagnetic radiation in anarrow wavelength band when exposed to radiation; and b) one or morelinking agents, each of said one or more linking agents linked to one ormore of said semiconductor nanocrystals.
 27. A semiconductor nanocrystalprobe capable of, in response to a first energy, providing a secondenergy, comprising: a) one or more semiconductor nanocrystal compounds;and b) one or more affinity molecules linked to said one or moresemiconductor nanocrystal compounds.
 28. The semiconductor nanocrystalprobe of claim 27 wherein said probe is capable of bonding with one ormore detectable substances.
 29. A semiconductor nanocrystal probecapable of bonding with one or more detectable substances and capableof, in response to exposure to a first energy, providing a secondenergy, said semiconductor nanocrystal probe comprising: a) one or moresemiconductor nanocrystals, each capable of, in response to exposure tosaid first energy, providing said second energy; b) one or more firstlinking agents, to which said one or more semiconductor nanocrystals arelinked, each of said one or more first linking agents capable of linkingto: i) one or more second linking agents; or ii) one or more affinitymolecules; and c) one or more affinity molecules linked either to saidone or more second linking agents or to said one or more first linkingagents, each of said one or more affinity molecules capable ofselectively bonding to said one or more detectable substances.
 30. Thesemiconductor nanocrystal probe of claim 29 wherein said one or moresemiconductor nanocrystals in said probe are capable of receiving saidfirst energy by fluorescence resonance energy transfer (FRET).
 31. Thesemiconductor nanocrystal probe of claim 29 wherein said one or moresemiconductor nanocrystals in said probe are capable of providing saidsecond energy by fluorescence resonance energy transfer (FRET).
 32. Thesemiconductor nanocrystal probe of claim 29 wherein said one or moresemiconductor nanocrystals in said probe are capable of receiving saidfirst energy by exposure to radiation.
 33. The semiconductor nanocrystalprobe of claim 32 wherein each of said one or more semiconductornanocrystals is capable of absorbing said radiation over a widebandwidth.
 34. The semiconductor nanocrystal probe of claim 29 whereinsaid second energy results from diffraction and/or scattering of saidfirst energy by at least one of said one or more semiconductornanocrystals.
 35. The semiconductor nanocrystal probe of claim 29wherein said second energy results from absorption of said first energyby at least one of said one or more semiconductor nanocrystals.
 36. Thesemiconductor nanocrystal probe of claim 29 wherein said semiconductornanocrystal probe is capable of transferring said second energy fromsaid semiconductor nanocrystal probe to one or more first proximalstructures.
 37. The semiconductor nanocrystal probe of claim 29 whereinsaid one or more semiconductor nanocrystals in said probe are capable ofproviding said second energy as electromagnetic radiation emitted bysaid one or more semiconductor nanocrystals.
 38. The semiconductornanocrystal probe of claim 29 wherein said one or more first linkingagents include a glass coating on said one or more semiconductornanocrystals, and said glass coating is capable of being linked to: i)said one or more second linking agents; or ii) said one or more affinitymolecules.
 39. The semiconductor nanocrystal probe of claim 38 whereinsaid glass coating on said one or more semiconductor nanocrystalscomprises a coating of silica glass.
 40. The semiconductor nanocrystalprobe of claim 29 wherein said one or more semiconductor nanocrystalscomprise two or more semiconductor nanocrystals wherein, in response toexposure to said first energy, said second energy provided by a first ofsaid two or more semiconductor nanocrystals is different than saidsecond energy provided by a second of said two or more semiconductornanocrystals.
 41. The semiconductor nanocrystal probe of claim 40wherein said two or more semiconductor nanocrystals comprise three ormore semiconductor nanocrystals wherein, in response to exposure to saidfirst energy, said second energy provided by a third of said three ormore semiconductor nanocrystals is different than said second energiesrespectively provided by said first and said second of said three ormore semiconductor nanocrystals.
 42. The semiconductor nanocrystal probeof claim 29 wherein said one or more semiconductor nanocrystals comprisetwo or more semiconductor nanocrystals wherein, in response to exposureto said first energy, said second energy provided by a first of said twoor more semiconductor nanocrystals is the same as said second energyprovided by a second of said two or more semiconductor nanocrystals. 43.The semiconductor nanocrystal probe of claim 42 wherein said two or moresemiconductor nanocrystals comprise three or more semiconductornanocrystals wherein, in response to exposure to said first energy, saidsecond energy provided by a third of said three or more semiconductornanocrystals is different than said second energies respectivelyprovided by said first and said second of said three or moresemiconductor nanocrystals.
 44. The semiconductor nanocrystal probe ofclaim 37 wherein said one or more semiconductor nanocrystals comprisetwo or more semiconductor nanocrystals wherein, in response to exposureto said first energy, said electromagnetic radiation emitted by a firstof said two or more semiconductor nanocrystals is different than saidelectromagnetic radiation emitted by a second of said two or moresemiconductor nanocrystals.
 45. The semiconductor nanocrystal probe ofclaim 44 wherein said two or more semiconductor nanocrystals comprisethree or more semiconductor nanocrystals wherein, in response toexposure to said first energy, said electromagnetic radiation emitted bya third of said three or more semiconductor nanocrystals is differentthan said electromagnetic radiation respectively emitted by said firstand said second of said three or more semiconductor nanocrystals. 46.The semiconductor nanocrystal probe of claim 37 wherein said one or moresemiconductor nanocrystals comprise two or more semiconductornanocrystals wherein, in response to exposure to said first energy, saidelectromagnetic radiation emitted by a first of said two or moresemiconductor nanocrystals is the same as said electromagnetic radiationemitted by a second of said two or more semiconductor nanocrystals. 47.The semiconductor nanocrystal probe of claim 46 wherein said two or moresemiconductor nanocrystals comprise three or more semiconductornanocrystals wherein, in response to exposure to said first energy, saidelectromagnetic radiation emitted by a third of said three or moresemiconductor nanocrystals is different than said electromagneticradiation respectively emitted by said first and said second of saidthree or more semiconductor nanocrystals.
 48. The semiconductornanocrystal probe of claim 29 wherein said semiconductor nanocrystalprobe is capable of treating a biological material to determine thepresence of said one or more detectable substances.
 49. Thesemiconductor nanocrystal probe of claim 29 wherein said semiconductornanocrystal probe is capable of treating an organic material todetermine the presence of said one or more detectable substances. 50.The semiconductor nanocrystal probe of claim 29 wherein saidsemiconductor nanocrystal probe is capable of treating an inorganicmaterial to determine the presence of said one or more detectablesubstances.
 51. The semiconductor nanocrystal probe of claim 29 whereinsaid semiconductor nanocrystal probe is capable of being exposed toelevated temperatures.
 52. The semiconductor nanocrystal probe of claim29 wherein said semiconductor nanocrystal probe is capable of beingexposed to temperatures up to about 150° C.
 53. The semiconductornanocrystal probe of claim 29 wherein said semiconductor nanocrystalprobe is capable of being exposed to temperatures up to about 100° C.54. The semiconductor nanocrystal probe of claim 29 wherein said one ormore affinity molecules are capable of being modified by one or moresynthetic steps to form a modified semiconductor nanocrystal probe. 55.The modified semiconductor nanocrystal probe of claim 54 wherein saidone or more affinity molecules comprise one or more strands of nucleicacid.
 56. The modified semiconductor nanocrystal probe of claim 55wherein said one or more strands of nucleic acid have been modified bynucleic acid synthesis to form said modified semiconductor nanocrystalprobe.
 57. The modified semiconductor nanocrystal probe of claim 56wherein said modified semiconductor nanocrystal probe is, prior to saidnucleic acid synthesis step, exposed to an elevated temperaturesufficient to cause said one or more strands of nucleic acid toseparate.
 58. The modified semiconductor nanocrystal probe of claim 57wherein said one or more strands of nucleic acid have been modified by apolymerase chain reaction.
 59. The semiconductor nanocrystal probe ofclaim 29 wherein at least one of said one or more semiconductornanocrystals comprise an alloy comprising two or more semiconductorsselected from the group consisting of Group III-V compounds, Group II-VIcompounds, Group IV elements, and combinations of same.
 60. Thesemiconductor nanocrystal probe of claim 29 wherein at least one of saidone or more semiconductor nanocrystals comprise: a) a core; and b) oneor more shells, concentrically disposed around the core.
 61. Thesemiconductor nanocrystal probe of claim 29 wherein said one or moreaffinity molecules comprise one or more first protein molecules, andsaid one or more detectable substances comprise one or more secondprotein molecules to which said one or more first protein moleculesbond.
 62. The semiconductor nanocrystal probe of claim 29 wherein saidone or more affinity molecules comprise one or more first smallmolecules, and said one or more detectable substances comprise one ormore second small molecules to which said one or more first smallmolecules bond.
 63. The semiconductor nanocrystal probe of claim 29wherein said one or more affinity molecules comprise one or more proteinmolecules, and said one or more detectable substances comprise one ormore small molecules to which said one or more protein molecules bond.64. The semiconductor nanocrystal probe of claim 29 wherein said one ormore affinity molecules comprise one or more small molecules, and saidone or more detectable substances comprise one or more protein moleculesto which said one or more small molecules bond.
 65. A semiconductornanocrystal probe capable of bonding with one or more detectablesubstances and capable of providing one or more detectable signals inresponse to exposure to radiation comprising: a) one or moresemiconductor nanocrystals, each capable of providing a detectablesignal in response to exposure to radiation; b) one or more firstlinking agents, each of said one or more first linking agents having afirst portion linked to at least one of said one or more semiconductornanocrystals, and each of said one or more first linking agents having asecond portion capable of linking to either one or more second linkingagents or to one or more affinity molecules; and c) one or more affinitymolecules linked either to said second linking agent or to said secondportion of said one or more first linking agents, each of said one ormore affinity molecules capable of selectively bonding to said one ormore detectable substances.
 66. The semiconductor nanocrystal probe ofclaim 65 wherein said one or more detectable signals provided by saidone or more semiconductor nanocrystals in response to said exposure toradiation comprises electromagnetic radiation emitted in a narrowwavelength band.
 67. A semiconductor nanocrystal probe capable ofbonding with one or more detectable substances and capable of, inresponse to exposure to a first energy, providing a second energy,comprising: a) one or more semiconductor nanocrystals, each capable of,in response to exposure to said first energy, providing said secondenergy; b) one or more first linking agents, at least one of said one ormore first linking agents comprising a three-dimensional shapedstructure capable of having linked thereto said one or moresemiconductor nanocrystals, each of said one or more first linkingagents capable of linking to: i) one or more second linking agents; orii) one or more affinity molecules; and c) one or more affinitymolecules linked either to said one or more second linking agents or tosaid one or more first linking agents, each of said one or more affinitymolecules capable of selectively bonding to said one or more detectablesubstances.
 68. The semiconductor nanocrystal probe of claim 67 whereinsaid three-dimensional shaped structure is capable of being linked, bycovalently bonding, to said one or more semiconductor nanocrystals. 69.The semiconductor nanocrystal probe of claim 67 wherein saidthree-dimensional shaped structure is capable of being linked, byadherence, to said one or more semiconductor nanocrystals.
 70. Thesemiconductor nanocrystal probe of claim 67 wherein saidthree-dimensional shaped structure is capable of being linked, byembedding, to said one or more semiconductor nanocrystals.
 71. Thesemiconductor nanocrystal probe of claim 67 wherein saidthree-dimensional shaped structure further comprises one or more organicmaterials.
 72. The semiconductor nanocrystal probe of claim 67 whereinsaid three-dimensional shaped structure further comprises one or moreinorganic materials.
 73. The semiconductor nanocrystal probe of claim 67wherein said three-dimensional shaped structure comprises a porous solidstructure which encapsulates said one or more semiconductornanocrystals.
 74. The semiconductor nanocrystal probe of claim 67wherein said three-dimensional shaped structure comprises a non-poroussolid structure which encapsulates said one or more semiconductornanocrystals.
 75. The semiconductor nanocrystal probe of claim 67wherein said three-dimensional shaped structure comprises a hollowstructure which encapsulates said one or more semiconductornanocrystals.
 76. The semiconductor nanocrystal probe of claim 67wherein said three-dimensional shaped structure comprises a sphericallyshaped structure.
 77. The semiconductor nanocrystal probe of claim 67wherein said three-dimensional shaped structure comprises two or moresubstructures wherein each substructure comprises one or more identicalsemiconductor nanocrystals.
 78. The semiconductor nanocrystal probe ofclaim 77 wherein said two or more substructures each comprise a onelayer in a layered structure.
 79. The semiconductor nanocrystal probe ofclaim 67 wherein said three-dimensional shaped structure comprises amedium transparent to: i) said first energy to which said one or moresemiconductor nanocrystals is exposed; and ii) said second energyprovided by said semiconductor nanocrystals in response to said exposureto said first energy.
 80. The semiconductor nanocrystal probe of claim67 wherein said three-dimensional shaped structure comprises a medium:i) capable of transferring said first energy to said one or moresemiconductor nanocrystals; and ii) transparent to said second energyprovided by said semiconductor nanocrystals in response to said exposureto said first energy.
 81. The semiconductor nanocrystal probe of claim67 wherein each of said one or more affinity molecules comprises amolecule of one or more strands of nucleic acid, and each of said one ormore detectable substances comprises a molecule of one or more strandsof nucleic acid with which said probe bonds.
 82. A process for forming asemiconductor nanocrystal compound capable of linking to one or moreaffinity molecules and capable of, in response to exposure to a firstenergy, providing a second energy, said semiconductor nanocrystalcompound, said process comprising linking together: a) one or moresemiconductor nanocrystals, each capable of, in response to exposure tosaid first energy, providing said second energy; and b) one or morelinking agents.
 83. The process for forming a semiconductor nanocrystalcompound of claim 82 including the steps of: a) linking said one or moresemiconductor nanocrystals to one or more first linking agents; and b)linking said one or more first linking agents to one or more secondlinking agents capable of linking to said one or more affinitymolecules.
 84. The process for forming a semiconductor nanocrystalcompound of claim 82 which further comprises the steps of: a) forming aglass coating on said one or more semiconductor nanocrystals; and b)treating said glass coating with one or more linking agents capable oflinking to said glass coating and also capable of linking to said one ormore affinity molecules.
 85. The process for forming a semiconductornanocrystal compound of claim 82 which further comprises the steps of:a) forming a glass coating, as a first linking agent, on said one ormore semiconductor nanocrystals; and b) treating said glass coating withone or more second linking agents capable of linking to said glasscoating and also capable of linking to said one or more affinitymolecules.
 86. The process for forming a semiconductor nanocrystalcompound of claim 82 wherein said step of linking said one or moresemiconductor nanocrystals to said one or more first linking agentsfurther comprises linking said one or more semiconductor nanocrystals toone or more three-dimensional shaped structures comprising said one ormore first linking agents.
 87. A process for forming a semiconductornanocrystal probe capable of, in response to a first energy, providing asecond energy, said process comprising linking together: a) one or moresemiconductor nanocrystal compounds; and b) one or more affinitymolecules.
 88. A process for forming a semiconductor nanocrystal probecapable of bonding with one or more detectable substances and capableof, in response to exposure to a first energy, providing a secondenergy, said process comprising the steps of: a) linking one or morefirst linking agents with one or more semiconductor nanocrystals, saidsemiconductor nanocrystals each capable of, in response to exposure tosaid first energy, providing said second energy; and b) linking said oneor more first linking agents to either: i) one or more second linkingagents; or ii) one or more affinity molecules capable of selectivelybonding to said one or more detectable substances; and c) linking saidone or more affinity molecules to said one or more second linking agentswhen said one or more first linking agents are linked to said one ormore second linking agents.
 89. The process for forming a semiconductornanocrystal probe of claim 88 wherein said step of linking together saidone or more semiconductor nanocrystals and said one or more firstlinking agents is carried out prior to said steps of: a) linking saidone or more first linking agents to either: i) said one or more secondlinking agents; or ii) said one or more affinity molecules capable ofselectively bonding to said one or more detectable substances; and b)linking said one or more affinity molecules to said one or more secondlinking agents when said one or more first linking agents are linked tosaid one or more second linking agents.
 90. The process for forming asemiconductor nanocrystal probe of claim 88 wherein said steps of: a)linking said one or more first linking agents to either: i) said one ormore second linking agents; or ii) said one or more affinity moleculescapable of selectively bonding to said one or more detectablesubstances; and b) linking said one or more affinity molecules to saidone or more second linking agents when said one or more first linkingagents are linked to said one or more second linking agents; are carriedout prior to said step of linking together said one or moresemiconductor nanocrystals and said one or more first linking agents.91. The process for forming a semiconductor nanocrystal probe of claim88 which further comprises the steps of: a) forming a glass coating, asa first linking agent, on said one or more semiconductor nanocrystals;and b) treating said glass coating with either: i) one or more of saidsecond linking agents which are capable of linking to said glasscoating; or ii) said one or more affinity molecules.
 92. The process forforming a semiconductor nanocrystal probe of claim 88 wherein said stepof linking said one or more semiconductor nanocrystals to said one ormore first linking agents further comprises linking said one or moresemiconductor nanocrystals to one or more three-dimensional shapedstructures comprising said one or more first linking agents.
 93. Theprocess for forming a semiconductor nanocrystal probe of claim 92wherein said three-dimensional shaped structure is formed by forming alayered structure having two or more layers.
 94. The process for forminga semiconductor nanocrystal probe of claim 88 wherein at least one ofsaid one or more affinity molecules comprises an affinity moleculecapable of being treated in a further step to form a modifiedsemiconductor nanocrystal probe.
 95. The process for forming a modifiedsemiconductor nanocrystal probe of claim 94 wherein said step of linkingsaid one or more affinity molecules to either one or more first linkingagents or one or more second linking agents further comprises linkingsaid first or second linking agents to one or more strands of nucleicacid which comprise said one or more affinity molecules.
 96. The processfor forming a modified semiconductor nanocrystal probe of claim 95comprising the further step of modifying said one or more strands ofnucleic acid by nucleic acid synthesis to form said modifiedsemiconductor nanocrystal probe.
 97. The process for forming a modifiedsemiconductor nanocrystal probe of claim 96 wherein: (a) each of saidone or more strands of nucleic acid comprises from 1 to about 50 nucleicacid monomers; and (b) said nucleic acid synthesis comprises theaddition of from 1 to about 500,000 nucleic acid monomers to said one ormore strands of nucleic acid.
 98. The process for forming a modifiedsemiconductor nanocrystal probe of claim 96 wherein said step ofmodifying said one or more strands of nucleic acid by said nucleic acidsynthesis further includes the step of exposing said semiconductornanocrystal probe to an elevated temperature sufficient to cause saidone or more strands of said nucleic acid to separate.
 99. The processfor forming a modified semiconductor nanocrystal probe of claim 98wherein said step of modifying said one or more strands of nucleic acidby nucleic acid synthesis further comprises modifying said one or morestrands of nucleic acid by a polymerase chain reaction.
 100. In aprocess wherein a precursor semiconductor nanocrystal probe has alreadybeen formed by linking one or more semiconductor nanocrystals with oneor more linking agents, and linking said one or more linking agents withone or more affinity molecules comprising one or more nucleic acidmonomers, the further step which comprises subjecting said precursorprobe to nucleic acid synthesis to form a modified semiconductornanocrystal probe.
 101. A process for treating a material by introducingone or more semiconductor nanocrystal probes into said material whichcomprises: a) contacting said material with one or more semiconductornanocrystal probes, said one or more semiconductor nanocrystal probeseach comprising: i) one or more semiconductor nanocrystals, each capableof, in response to exposure to a first energy, providing a secondenergy; ii) one or more first linking agents, to which said one or moresemiconductor nanocrystals are linked, each of said one or more firstlinking agents capable of linking to: 1) one or more second linkingagents; or 2) one or more affinity molecules; and iii) one or moreaffinity molecules linked either to said one or more second linkingagents or to said one or more first linking agents; b) exposing said oneor more semiconductor nanocrystal probes in said material to said firstenergy whereby said second energy is provided by said one or moresemiconductor nanocrystals in said one or more semiconductor nanocrystalprobes.
 102. The process for treating a material of claim 101 whereinsaid one or more semiconductor nanocrystal probes are capable of bondingto one or more detectable substances in said material, and said secondenergy provided by said one or more semiconductor nanocrystal probes isindicative of the presence of said one or more detectable substances, insaid material, bonded to said one or more semiconductor nanocrystalprobes.
 103. The process for treating a material of claim 101 whereinsaid one or more semiconductor nanocrystal probes are capable oftransferring said second energy to one or more first proximalstructures; and said process includes the further step of transferringsaid second energy from said one or more semiconductor nanocrystalprobes to said one or more first proximal structures.
 104. The processfor treating a material of claim 103 comprising the further step ofdetecting a detectable signal provided by said one or more firstproximal structures in response to said second energy transferred fromsaid one or more semiconductor nanocrystal probes.
 105. The process fortreating a material of claim 104 wherein said one or more semiconductornanocrystal probes transfer said second energy to said one or more firstproximal structures by way of a non-radiative pathway.
 106. The processfor treating a material of claim 103 wherein at least one of said one ormore first proximal structures undergoes a chemical change in responseto said second energy transferred from said one or more semiconductornanocrystal probes to said one or more first proximal structures. 107.The process for treating a material of claim 106 wherein said chemicalchange comprises a photolytic cleavage of one or more covalent bonds.108. The process for treating a material of claim 106 wherein said firstenergy to which said semiconductor nanocrystal probe is exposedcomprises radiation having a first wavelength, and said second energytransferred from said one or more semiconductor nanocrystal probes tosaid one or more first proximal structures comprises radiation having asecond wavelength, wherein said second wavelength is shorter than saidfirst wavelength.
 109. The process for treating a material of claim 108whereby said first energy having said first wavelength is converted tosaid second energy having said second shorter wavelength through aprocess of two-photon absorption.
 110. The process for treating amaterial of claim 108 wherein said first energy to which saidsemiconductor nanocrystal probe is exposed comprises infrared radiation,and wherein said second energy transferred from said one or moresemiconductor nanocrystal probes to said one or more first proximalstructures comprises ultraviolet radiation.
 111. The process fortreating a material of claim 103 wherein at least one of said one ormore first proximal structures undergoes conformational changes inresponse to said second energy transferred from said one or moresemiconductor nanocrystal probes to said one or more first proximalstructures.
 112. The process for treating a material of claim 103wherein said second energy transferred from said one or moresemiconductor nanocrystal probes to said one or more first proximalstructures is heat energy.
 113. The process for treating a material ofclaim 103 wherein said second energy transferred from said one or moresemiconductor nanocrystal probes to said one or more first proximalstructures, is transferred from said one or more first proximalstructures to one or more second proximal structures.
 114. A process fortreating a material using one or more semiconductor nanocrystal probesto determine the presence of one or more detectable substances in saidmaterial which comprises: a) contacting said material with one or moresemiconductor nanocrystal probes, said one or more semiconductornanocrystal probes each comprising: i) one or more semiconductornanocrystals, each capable of, in response to exposure to a firstenergy, providing a second energy; ii) one or more first linking agents,to which said one or more semiconductor nanocrystals are linked, each ofsaid one or more first linking agents capable of linking to: 1) one ormore second linking agents; or 2) one or more affinity molecules; andiii) one or more affinity molecules linked either to said one or moresecond linking agents or to said one or more first linking agents, eachof said one or more affinity molecules capable of selectively bonding tosaid one or more detectable substances; b) exposing said one or moresemiconductor nanocrystal probes to said first energy; and c) detectingsaid second energy provided by said one or more semiconductornanocrystals in said one or more semiconductor nanocrystal probes bondedto said one or more detectable substances in said material.
 115. Theprocess for treating a material of claim 114 further including theoptional step of removing from said material any of said one or moresemiconductor nanocrystal probes not bonded to said one or moredetectable substances in said material prior to said step of detectingsaid second energy provided by any of said one or more probes bonded tosaid one or more detectable substances.
 116. The process for treating amaterial of claim 115 wherein said one or more detectable substances,the presence of which is being determined, comprise a biologicalmaterial.
 117. The process for treating a material of claim 115 whereinsaid one or more detectable substances are present on the surface orinterior of biological cells.
 118. The process for treating a materialof claim 117 wherein two or more of said semiconductor nanocrystalprobes are bonded to said one or more detectable substances in saidmaterial, each of said two or more probes capable of providing a secondenergy comprising a detectable signal in response to exposure to saidfirst energy, and at least two of said detectable signals from said twoor more probes are simultaneously detected.
 119. The process fortreating a material of claim 117 wherein said material is flowed throughone or more compartments transparent to: a) said first energy to whichsaid material is exposed; and b) said second energy provided by said oneor more semiconductor nanocrystal probes in response to exposure to saidfirst energy.
 120. The process for treating a material of claim 119wherein two or more of said semiconductor nanocrystal probes are bondedto said one or more detectable substances in said material, each of saidtwo or more probes capable of providing a second energy comprising adetectable signal in response to exposure to said first energy, and atleast two of said detectable signals from said two or more probes areconsecutively detected.
 121. The process for treating a material ofclaim 119 wherein two or more of said semiconductor nanocrystal probesare bonded to said one or more detectable substances in said material,each of said two or more probes capable of providing a second energycomprising a detectable signal in response to exposure to said firstenergy, and at least two of said detectable signals from said two ormore probes are simultaneously detected.
 122. The process for treating amaterial of claim 115 wherein said one or more affinity molecules hasbeen modified with an organic substance prior to said treating of saidmaterial.
 123. The process for treating a material of claim 115 whereinsaid one or more affinity molecules comprise one or more strands ofnucleic acid, and said one or more detectable substances in saidmaterial also comprise one or more strands of nucleic acid; and saidstep of contacting said material with said one or more probes causessaid one or more strands of nucleic acid of said one or more probes tobond to said one or more strands of nucleic acid in said material bynucleic acid hybridization.
 124. The process for treating a material ofclaim 115 wherein said first energy is transferred from one or moreproximal sources to said one or more semiconductor nanocrystal probes.125. The process for treating a material of claim 124 wherein said oneor more proximal sources transfer said first energy to said one or moresemiconductor nanocrystal probes by way of a radiative pathway.
 126. Theprocess for treating a material of claim 124 wherein said one or moreproximal sources transfer said first energy to said one or moresemiconductor nanocrystal probes by way of a non-radiative pathway. 127.The process for treating a material of claim 124 wherein said secondenergy indicates the concentration of at least one of said one or moreproximal sources.
 128. The process for treating a material of claim 124wherein said second energy indicates the distance between at least oneof said one or more proximal sources and at least one of said one ormore semiconductor nanocrystal probes.
 129. The process for treating amaterial of claim 124 wherein said second energy indicates an eventwhich causes said one or more proximal sources to be spatially proximalto said one or more semiconductor nanocrystal probes.
 130. The processfor treating a material of claim 124 wherein said one or more proximalsources undergo nuclear decay; and said first energy to which said oneor more semiconductor nanocrystals are exposed, comprises radiationoriginating from said one or more proximal sources.
 131. The process fortreating a material of claim 124 wherein said first energy to which saidone or more semiconductor nanocrystals are exposed, is transmittedthrough said one or more proximal sources from an energy source separatefrom said one or more proximal sources.
 132. The process for treating amaterial of claim 115 wherein at least one of said one or more firstlinking agents comprises a three dimensional structure.
 133. The processfor treating a material of claim 132 wherein said three dimensionalstructure is linked to two or more of said semiconductor nanocrystals.134. The process for treating a material of claim 133 wherein saidsecond energy provided by said two or more semiconductor nanocrystalslinked to said three dimensional structure comprises one or moredetectable signals.
 135. The process for treating a material of claim115 wherein said one or more semiconductor nanocrystal probes comprisetwo or more semiconductor nanocrystal probes; and wherein, in responseto exposure to said first energy, said second energy provided by a firstof said two or more semiconductor nanocrystal probes is different thansaid second energy provided by a second of said two or moresemiconductor nanocrystal probes.
 136. The process for treating amaterial of claim 135 wherein said two or more semiconductor nanocrystalprobes comprise three or more semiconductor nanocrystal probes wherein,in response to exposure to said first energy, said second energyprovided by a third of said three or more semiconductor nanocrystalprobes is different than said second energies respectively provided bysaid first and said second of said three or more semiconductornanocrystal probes.
 137. The process for treating a material of claim115 wherein said one or more semiconductor nanocrystal probes comprisetwo or more semiconductor nanocrystal probes wherein, in response toexposure to said first energy, said second energy provided by a first ofsaid two or more semiconductor nanocrystal probes is the same as saidsecond energy provided by a second of said two or more semiconductornanocrystal probes.
 138. The process for treating a material of claim137 wherein said two or more semiconductor nanocrystal probes comprisethree or more semiconductor nanocrystal probes wherein, in response toexposure to said first energy, said second energy provided by a third ofsaid three or more semiconductor nanocrystal probes is different thansaid second energies respectively provided by said first and said secondof said three or more semiconductor nanocrystal probes.
 139. A processfor treating a material to determine the presence of one or moredetectable substances in said material which comprises: a) contactingsaid material with one or more semiconductor nanocrystal probes capableof bonding with said one or more detectable substances, if present, insaid material, and capable of providing one or more detectable signalsin response to exposure to energy, said one or more semiconductornanocrystal probes comprising: i) one or more semiconductor nanocrystalseach capable of providing a detectable signal in response to exposure toenergy; ii) one or more first linking agents, to which said one or moresemiconductor nanocrystals are linked, each of said one or more firstlinking agents capable of linking to: 1) one or more second linkingagents; or 2) one or more affinity molecules; and iii) one or moreaffinity molecules linked either to said one or more second linkingagents or to said one or more first linking agents, each of said one ormore affinity molecules capable of selectively bonding to said one ormore detectable substances; b) optionally removing, from said material,any of said semiconductor nanocrystal probes not bonded to said one ormore detectable substances; and c) exposing said material to energycapable of causing said one or more semiconductor nanocrystals toprovide one or more detectable signals in response to said energy,indicative of the presence of said one or more detectable substances insaid material; and d) detecting said one or more detectable signalsprovided by said one or more semiconductor nanocrystals in said one ormore semiconductor nanocrystal probes.
 140. The process for treating amaterial of claim 139 wherein said step of exposing said material toenergy capable of causing said one or more semiconductor nanocrystals toprovide one or more detectable signals further comprises exposing saidmaterial to a source of radiation.
 141. The process for treating amaterial of claim 140 wherein said source of radiation comprises asource of electromagnetic radiation.
 142. The process for treating amaterial of claim 141 wherein said source of electromagnetic radiationis capable of emitting electromagnetic radiation of a broad or narrowwavelength band.
 143. The process for treating a material of claim 142wherein said broad or narrow wavelength band of electromagneticradiation comprises electromagnetic radiation selected from the groupconsisting of visible light, ultraviolet light, x-rays, and infraredlight.
 144. The process for treating a material of claim 140 whereinsaid source of radiation comprises a particle beam.
 145. The process fortreating a material of claim 139 wherein said one or more detectablesignals result from diffraction and/or scattering of said energy by atleast one of said one or more semiconductor nanocrystals.
 146. Theprocess for treating a material of claim 145 wherein said step ofexposing said material to energy capable of providing said one or moredetectable signals from diffraction and/or scattering of said energyfurther comprises exposing said material to a particle beam.
 147. Theprocess for treating a material of claim 145 wherein: a) said step ofexposing said materials to energy capable of causing said one or moresemiconductor nanocrystals to scatter or diffract energy; and b) saidstep of detecting said one or more detectable signals resulting fromsaid scattering or diffraction of energy; are both carried out using atransmission electron microscope.
 148. The process for treating amaterial of claim 145 wherein: a) said step of exposing said materialsto energy capable of causing said one or more semiconductor nanocrystalsto scatter or diffract energy; and b) said step of detecting said one ormore detectable signals resulting from said scattering or diffraction ofenergy; are both carried out using a scanning electron microscope. 149.The process for treating a material of claim 139 wherein said one ormore detectable signals result from absorption of said energy by atleast one of said one or more semiconductor nanocrystals.
 150. Theprocess for treating a material of claim 139 wherein said one or moresemiconductor nanocrystals in said one or more probes are capable ofproviding said one or more detectable signals as electromagneticradiation emitted by said one or more semiconductor nanocrystals. 151.The process for treating a material of claim 150 wherein said one ormore semiconductor nanocrystals are capable of emitting electromagneticradiation in a narrow wavelength band when exposed to said energy. 152.The process for treating a material of claim 150 wherein saidelectromagnetic radiation emitted by said one or more semiconductornanocrystals comprises visible light.
 153. The process for treating amaterial of claim 150 wherein said electromagnetic radiation emitted bysaid one or more semiconductor nanocrystals comprises ultraviolet light.154. The process for treating a material of claim 150 wherein saidelectromagnetic radiation emitted by said one or more semiconductornanocrystals comprises infrared light.
 155. The process for treating amaterial of claim 139 wherein said energy to which said one or moresemiconductor nanocrystals are exposed comprises electromagneticradiation of a broad wavelength band; and said one or more detectablesignals, provided by said one or more semiconductor nanocrystals inresponse to said exposure, comprise electromagnetic radiation emitted ina narrow wavelength band.